Recombinant ROBO2 proteins, compositions, methods and uses thereof

ABSTRACT

The invention provides recombinant Roundabout Receptor 2 (ROBO2) proteins designed to bind SLIT ligands and prevent their binding to ROBO2 cell surface receptors. Also provided are methods for use of these recombinant ROBO2 proteins.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Nos.62/514,242, filed Jun. 2, 2017; and 62/663,082, filed Apr. 26, 2018,which are hereby incorporated by reference here in their entirety.

PARTIES TO A JOINT RESEARCH STATEMENT

The presently claimed invention was made by or on behalf of the belowlisted parties to a joint research agreement. The joint researchagreement was in effect on or before the date the claimed invention wasmade and the claimed invention was made as a result of activitiesundertaken within the scope of the joint research agreement. The partiesto the joint research agreement are BOSTON MEDICAL CENTER CORP. andPFIZER INC.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 3, 2018, isnamed PCFC-0043-101-SL.txt and is 54,386 bytes in size.

BACKGROUND

Chronic kidney disease (CKD) is a worldwide public health problem, whichoften leads to end-stage renal failure. CKD affects an estimated 13% ofthe population or ˜27 million in the United States and over 500 millionpeople worldwide. The prevalence of CKD is predicted to continue toincrease because of the ongoing epidemic of diabetes and obesity withinthe general population. About half a million CKD patients in the US (˜7million worldwide) will progress to end-stage renal disease (ESRD) andneed dialysis or kidney transplantation for survival. The morbidity andmortality of ESRD are high and cost the US at least $40 billion eachyear. Proteinuria (i.e., the presence of an excess of serum proteins inthe urine—commonly defined as urine albumin level>30 mg/day) is an earlybiomarker, risk factor and surrogate outcome of CKD in patients with andwithout diabetes. Treatment to reduce the level of proteinuria duringearly stages of CKD can slow progression to ESRD. However, there is nokidney podocyte specific anti-proteinuric treatment currently availablefor CKD patients with proteinuria.

Podocytes are specialized epithelial cells that extend primary andsecondary processes to cover the outer surface of the glomerularbasement membrane. The actin-rich interdigitating secondary processes(i.e., foot processes) from neighboring podocytes create filtrationslits bridged by a semi-porous slit-diaphragm that forms the finalbarrier to protein permeation. Proteinuria is the clinical signature ofpodocyte injury in diabetic and non-diabetic kidney disease. There is anexpanding group of published studies showing that hereditary,congenital, or acquired abnormalities in the molecular component ofpodocytes leads to proteinuria. Whereas genetic mutations of podocyteslit-diaphragm proteins such as nephrin and podocin are associated withhereditary forms of proteinuric kidney disease, it has becomeincreasingly evident that the proteins that make up and associate withthe slit-diaphragm are more than a simple structural barrier. Thus,substantial evidence suggests that these proteins form a balancedsignaling network that may influence podocyte foot process structure andfunction through interaction with the actin cytoskeleton.

Roundabout (ROBO) Receptors

Roundabout Receptor 2 (ROBO2, also referred to as Roundabout GuidanceReceptor 2 or Roundabout homolog 2) is a receptor for Slit GuidanceLigand (SLIT) protein ligands. ROBO2 is expressed at the basal surfaceof glomerular podocytes in the kidney and Slit Guidance Ligand 2 (SLIT2)is present in kidney glomeruli. Upon SLIT2 binding, ROBO2 forms acomplex with nephrin in the glomerular filtration barrier and acts as anegative regulator to inhibit nephrin-induced actin polymerization. Theloss of ROBO2 increases the actin polymerization in the podocyte andalleviates the abnormal podocyte structural phenotype found innephrin-null mice. Loss of ROBO2 also increases adhesion of podocytes tothe glomerular basement membrane in mice. These data, along with theobservation that a patient with ROBO2 chromosomal translocation lacksproteinuria, suggests that blocking of SLIT2-ROBO2 signaling pathwaycould increase nephrin-induced actin polymerization to reduceproteinuria. Blocking of ROBO2 signaling may also restore glomerularfiltration barrier in proteinuric disease by up-regulation of nephrininduced actin polymerization.

SLIT1, SLIT2 and SLIT3. SLITs are secreted proteins associated with theextracellular matrix. The protein sequence of all SLITs shows a highdegree of conservation and have the same structure: an N-terminus signalpeptide; four tandem leucine-rich repeat domains (LRR) termed D1-D4; sixepidermal growth factor (EGF)-like domains; a laminin G-like domain; afurther one (invertebrates) or three (vertebrates) EGF-like domains anda C terminal cysteine knot domain. SLIT ligands can be cleaved to yielda short C-terminus fragment of unknown function (SLIT-C product) and along N-terminus fragment (SLIT-N product) that is active and mediatesbinding to ROBOs. SLIT ligands, as well as cleavage products (e.g.,SLIT-N, SLIT2-D2) described herein can be used to assess ROBO2 activity.

Four ROBO receptors have been characterized in vertebrates: ROBO1/Dutt1;ROBO2; ROBO3/Rig-1 and ROBO4/Magic Roundabout. ROBO1, ROBO2 and ROBO3share a common extracellular domain (ECD) structure that is reminiscentof cell adhesion molecules. This region contains fiveimmunoglobulin-like (Ig-like) domains (Ig1, Ig2, Ig3, Ig4 and Ig5)followed by three fibronectin type 3 (FN3) repeats (FIG. 1A). Inaddition, ROBO2 has four cytoplasmic conserved (CC) sequences in itsintracellular domain as illustrated in FIG. 1A.

The sequence of full length human ROBO2 precursor is shown as SEQ ID NO:24. A 21 amino acid ROBO2 leader sequence (SEQ ID NO: 17; residues 1-21according to the numbering set forth in SEQ ID NO: 24) is cleaved duringprotein production to produce mature ROBO2 (FIG. 1A). Residues 22-859according to the numbering set forth in SEQ ID NO: 24 form theextracellular domain, residues 860-880 set forth in SEQ ID NO: 24 formthe transmembrane domain, and residues 881-1378 set forth in SEQ ID NO:24 form the cytoplasmic domain (FIG. 1A).

Exemplary sequences of the five Ig-like domains (Ig1, Ig2, Ig3, Ig4 andIg5) of ROBO2 are shown in Table 23. The ROBO2 pre-Ig1sequence (SEQ IDNO: 8), the Ig1-Ig2 inter-domain linker (SEQ ID NO: 10) and the Ig2-Ig3inter-domain linker (SEQ ID NO: 12) are also disclosed in Table 23.

The D2 LRR domain of the SLITs and Ig1 and Ig2 domains of the ROBOs areevolutionary conserved and are involved in binding. Ig1 and Ig2 domainsof ROBO together are also referred to as SLIT-binding domain. Studieshave shown that while both immunoglobulin-like (Ig-like) domains 1 and 2(Ig1 and Ig2) of ROBO2 interact with SLIT; the first Ig-like domain(Ig1) is the primary binding site for SLIT. In addition, previousstudies have indicated that removing the three fibronectin type III(FNIII) repeats has a greater negative effect on ROBO binding to SLITthan removal of the third and fourth immunoglobulin-like domains (Ig3and Ig4) (see, e.g., Liu et al., 2004, Molecular Cellular Neuroanatomy39:256-261).

Upon ROBO-SLIT binding, Rho GTPases and their regulators (GAPs and GEFs)are involved in the downstream signaling pathway. In the presence ofSLIT, SLIT-ROBO Rho GTPase activating protein 1 (srGAP1) binds to theCC3 domain of ROBO and inactivates RhoA and Cdc42. These effectorproteins are able to mediate, among other outcomes, repulsion, controlof cytoskeletal dynamics and cell polarity. In the presence of SLIT,Vilse/CrossGAP can also bind to the CC2 domain of ROBO and inhibit Rac1and Cdc42. Rac1 is also activated by the recruitment of the GEF proteinSon of sevenless (Sos) via the adaptor protein Dreadlocks (Dock), whichbinds to the CC2-3 domain of ROBO. This activates the downstream targetof Rac1 and p21-activated kinase (Pak), which also binds to ROBO CC2-3domains. These downstream signaling partners of ROBO control repulsionand cytoskeletal dynamics. The tyrosine kinase Abelson (Abl) can alsobind ROBO CC3 domain and antagonizes ROBO signaling throughphosphorylation of the CC1 domain and mediates cell adhesion. Enabled(Ena), a substrate of Abl, also binds ROBO CC1 and CC2 domains. Allthese downstream ROBO-SLIT molecules may be used to assess ROBO2activity, as well as to assess any neutralizing effect of a novelrecombinant ROBO2 protein disclosed herein.

In the kidney, ROBO2 forms a complex with nephrin through adaptorprotein Nck. In contrast to the role of nephrin that promotes actinpolymerization, SLIT-ROBO2 signaling inhibits nephrin-induced actinpolymerization. Thus, the binding of ROBO2 intracellular domain and Nckmay be used to assess ROBO2 activity.

Patients suffering from many glomerular diseases (including FocalSegmental Glomerular Sclerosis) currently have no therapies available topreserve renal function or otherwise treat the disease. Further, thereis no treatment currently available for CKD patients with proteinuria.Accordingly, there is a need for developing a therapeutic that modulatesROBO2-SLIT signaling, thereby preserving or modulating podocytefunctions and reducing proteinuria or otherwise treating or preventing arenal disease associated with or mediated by ROBO2-SLIT binding andsignaling.

SUMMARY OF THE INVENTION

The invention provides recombinant ROBO2 proteins that bind to SLITligands, as well as uses, and associated methods thereof. Those skilledin the art will recognize, or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe invention described herein. Such equivalents are intended to beencompassed by the following embodiments (E).

E1. A recombinant Roundabout Receptor 2 (ROBO2) protein comprising aminoacid residues 1 to 203 according to the numbering of SEQ ID NO: 1, andfurther comprising an immunoglobulin heavy chain constant domain.

E2. A recombinant Roundabout Receptor 2 (ROBO2) protein consistingessentially of amino acid residues 1 to 203 according to the numberingof SEQ ID NO: 1, and an immunoglobulin heavy chain constant domain.

E3. A recombinant Roundabout Receptor 2 (ROBO2) protein comprising (i) aSLIT-binding moiety; and (ii) a half-life extending moiety, wherein saidSLIT-binding moiety comprises a portion of the ROBO2 extracellulardomain.

E4. The recombinant ROBO2 protein of E3, wherein said portion of saidROBO2 extracellular domain comprises the first two immunoglobulin-like(Ig1 and Ig2) domains of ROBO2 and a C-terminus sequence consisting ofthe sequence of SEQ ID NO: 12.

E5. The recombinant ROBO2 protein of E3, wherein said portion of saidROBO2 extracellular domain consists essentially of ROBO2pre-immunoglobulin-like 1 (Ig1) sequence (SRLRQEDFP (SEQ ID NO: 8),first immunoglobulin-like domain (Ig1), inter-domain linker betweenfirst and second immunoglobulin-like domains (Ig1-Ig2 inter-domainlinker; VALLR (SEQ ID NO: 10)), second immunoglobulin-like domain (Ig2),and inter-domain linker between second and third immunoglobulin-likedomains (Ig2-Ig3 inter-domain linker; VFER (SEQ ID NO: 12)).

E6. The recombinant ROBO2 protein of any one of E3-E5, wherein saidportion of said ROBO2 extracellular domain consists essentially of aminoacid residues 1 to 203 according to the numbering of SEQ ID NO: 1.

E7. The recombinant ROBO2 protein of any one of E3-E6, wherein saidhalf-life extending moiety comprises an immunoglobulin domain.

E8. The recombinant ROBO2 protein of any one of E1, E2 or E7, whereinsaid immunoglobulin domain is an Fc domain of an IgA₁ IgA₂, IgD, IgE,IgM, IgG₁, IgG₂, IgG₃, or IgG₄.

E9. The recombinant ROBO2 protein of E8, wherein said Fc domain is theFc domain of human IgG₁.

E10. The recombinant ROBO2 protein of E9, wherein said Fc domain ismodified to eliminate effector function.

E11. The recombinant ROBO2 protein of E10, wherein said Fc domain is theFc domain of human IgG₁, and wherein said human IgG1 Fc domain comprisesat least one mutation selected from the group consisting of asubstitution from leucine to alanine at amino acid residue number 234(L234A), a substitution from leucine to alanine at amino acid residuenumber 235 (L235A), and a substitution from glycine to alanine at aminoacid residue number 237 (G237A) all according to the Eu numbering as setforth in Kabat.

E12. The recombinant ROBO2 protein of E9-E11, wherein said human IgG1 Fcdomain does not comprise a lysine at residue number 447 according to theEu numbering as set forth in Kabat.

E13. The recombinant ROBO2 protein of any one of E11-E12, wherein saidFc domain comprises amino acid residues 210 to 440 according to thenumbering of SEQ ID NO: 1.

E14. The recombinant ROBO2 protein of any one of E11-E12, wherein saidFc domain consists of amino acid residues 210 to 440 according to thenumbering of SEQ ID NO: 1.

E15. The recombinant ROBO2 protein of any one of E1, E2 or E7, whereinsaid amino acid residues 1 to 203 according to the numbering of SEQ IDNO: 1 are contiguous with said immunoglobulin domain.

E16. The recombinant ROBO2 protein of any one of E1, E2 or E7, whereinsaid amino acid residues 1 to 203 according to the numbering of SEQ IDNO: 1 are connected via a linker to said immunoglobulin domain.

E17. The recombinant ROBO2 protein of E16, wherein said linker is apeptidyl linker comprising from about 1 to 30 amino acid residues.

E18. The recombinant ROBO2 protein of E17, wherein said peptidyl linkeris selected from the group consisting of:

a) a glycine rich peptide;

b) a peptide comprising glycine and serine;

c) a peptide having a sequence (Gly-Gly-Ser)_(n), wherein n is 1, 2, 3,4, 5, or 6 (SEQ ID NO: 22); and

d) a peptide having a sequence (Gly-Gly-Gly-Gly-Ser)_(n), wherein n is1, 2, 3, 4, 5, or 6 (SEQ ID NO: 23).

E19. The recombinant ROBO2 protein of E18, wherein said peptidyl linkeris (Gly-Gly-Ser)₂ (SEQ ID NO: 15).

E20. A recombinant ROBO2-Fc protein comprising the amino acid sequenceof SEQ ID NO: 1.

E21. A recombinant ROBO2-Fc protein consisting of the amino acidsequence of SEQ ID NO: 1.

E22. A recombinant ROBO2-Fc protein comprising an amino acid sequence atleast 90% identical to the amino acid sequence of SEQ ID NO: 1.

E23. The recombinant ROBO2 protein of any one of E1-E22, comprising theamino acid sequence encoded by the insert of the plasmid deposited atthe ATCC and having ATCC Accession No. PTA-124008.

E24. The recombinant ROBO2-Fc protein of any one of E4 or E5, whereinsaid Ig1 of ROBO2 comprises at least one of the following mutations:S17T and R73Y, each numbered according to SEQ ID NO: 1.

E25. A recombinant ROBO2-Fc protein comprising the amino acid sequenceof SEQ ID NO: 19.

E26. The recombinant ROBO2-Fc protein of E25, wherein the protein doesnot comprise a C-terminal lysine located at amino acid residue number441 according to the numbering of SEQ ID NO: 19.

E27. The recombinant ROBO2 protein of any one of E1-E26, wherein saidROBO2 is human ROBO2.

E28. The recombinant ROBO2 protein of any one of E1-E27, wherein saidprotein binds SLIT2 with a binding affinity (K_(D)) of or less than:about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300pM, about 250 pM, about 200 pM, about 150 pM, about 100 pM, about 50 pM,about 40 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, about10 pM, about 5 pM, or about 1 pM.

E29. The recombinant ROBO2 protein of any one of E1-E27, wherein saidprotein binds SLIT2 with a K_(D) that is at least about 2-fold, about4-fold, about 6-fold, about 8-fold, about 10-fold, about 20-fold, about40-fold, about 60-fold, about 80-fold, about 100-fold, about 120-fold,about 140-fold, about 160-fold, lower than the K_(D) value for bindingof ROBO1 to SLIT2.

E30. The recombinant ROBO2 protein of any one of E1-E29, wherein saidprotein binds SLIT2 with a K_(D) value that is at least about 2-fold,about 4-fold, about 6-fold, about 8-fold, about 10-fold, about 20-fold,about 40-fold, about 60-fold, about 80-fold, about 100-fold, about120-fold, about 140-fold, about 160-fold, lower than the K_(D) value forbinding of a ROBO1-Fc protein to SLIT2.

E31. The recombinant ROBO2 protein of any one of E28-E30, wherein saidK_(D) is measured by surface plasmon resonance (SPR).

E32. The recombinant ROBO2 protein of E31, wherein said K_(D) ismeasured using a Biacore T200 instrument.

E33. The recombinant ROBO2 protein of any one of E28-E30, wherein saidK_(D) is measured by bio-layer interferometry (BLI).

E34. The recombinant ROBO2 protein of E33, wherein said K_(D) ismeasured using a ForteBio Octet instrument.

E35. The recombinant ROBO2 protein of any one of E1-E34, wherein saidprotein inhibits binding of a SLIT ligand and ROBO2.

E36. The recombinant ROBO2 protein of any one of E1-E34, wherein saidprotein inhibits ROBO2-dependent SLITx-N activity.

E37. The recombinant ROBO2 protein of any one of E1-E36, wherein saidprotein inhibits binding of a SLIT ligand and ROBO2 and inhibitsROBO2-dependent SLIT-N activity.

E38. The recombinant ROBO2 protein of any one of E1-E37, wherein saidROBO2-dependent SLITx-N activity is selected from the group consistingof actin polymerization, podocyte adhesion, and inhibition of neuronalcell migration.

E39. The recombinant ROBO2 protein of any one of E1-E38, wherein saidprotein has a half maximal inhibitory concentration (IC₅₀) of not morethan about 15 nM, about 13 nM, about 11 nM, about 9 nM, about 7 nM,about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM.

E40. The recombinant ROBO2 protein of E39, wherein said IC₅₀ is measuredby a homogenous time-resolved fluorescence (HTRF) assay for inhibitionof binding of ROBO2 to SLIT2.

E41. The recombinant ROBO2 protein of any one of E1-E40, wherein saidprotein has a half maximal IC₅₀ of not more than about 75 nM, about 65nM, about 55 nM, about 45 nM, about 35 nM, about 25 nM, about 15 nM,about 5 nM.

E42. The recombinant ROBO2 protein of E41, wherein said IC₅₀ is assessedby measuring SLIT2-N mediated inhibition of neuronal cell migration.

E43. The recombinant ROBO2 protein of any one of E28-E42, wherein saidSLIT2 is human SLIT2.

E44. The recombinant ROBO2 protein of any one of E1-E43, wherein two ofsaid recombinant ROBO2 proteins associate to form a homodimer.

E45. An isolated nucleic acid molecule encoding the recombinant ROBO2protein of any one of E1-E44.

E46. The isolated nucleic acid molecule of E45 comprising the nucleicacid sequence of SEQ ID NO: 21.

E47. The isolated nucleic acid molecule of E45 consisting of the nucleicacid sequence of SEQ ID NO: 21.

E48. An isolated nucleic acid comprising the nucleic acid sequence ofthe insert of the plasmid deposited at the ATCC and having ATCCAccession No. PTA-124008.

E49. A recombinant ROBO2 protein comprising an amino acid sequenceencoded by the sequence of SEQ ID NO: 21.

E50. A recombinant ROBO2 protein comprising an amino acid sequenceencoded by a sequence that is at least 85%, 90%, 95%, or 99% identicalto the sequence of SEQ ID NO:

E51. A recombinant ROBO2 protein comprising an amino acid sequenceencoded by a sequence capable of hybridizing under highly stringentconditions to the sequence of SEQ ID NO: 21.

E52. A vector comprising the nucleic acid molecule of any one ofE45-E48.

E53. A host cell comprising the nucleic acid molecule of any one ofE45-E48.

E54. A host cell comprising the vector of E52.

E55. The host cell of E53 or E54, wherein said cell is a mammalian cell.

E56. The host cell of E53 or E54, wherein said host cell is a CHO cell,a HEK-293 cell, or a Sp2.0 cell.

E57. A method of making a recombinant ROBO2 protein, comprisingculturing the host cell of any one of E53-E56 under conditions whereinsaid recombinant ROBO2 protein is expressed.

E58. The method of E57, further comprising isolating said recombinantROBO2 protein.

E59. A pharmaceutical composition comprising a recombinant ROBO2 proteinof any one of E1-E44, and a pharmaceutically acceptable carrier orexcipient.

E60. A method of reducing the biological activity of ROBO2, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59.

E61. The method of E60, wherein said biological activity of ROBO2 isselected from the group consisting of binding to at least one SLITligand, actin polymerization, podocyte adhesion, inhibitingSLIT2-N-mediated inhibition of neuronal cell migration, binding of ROBO2with srGAP1, and binding of ROBO2 with Nck.

E62. A method of treating renal disease, comprising administering to asubject in need thereof a therapeutically effective amount of therecombinant ROBO2 protein of any one of E1-E44, or the pharmaceuticalcomposition of E59.

E63. A method of preserving podocyte function, comprising contactingsaid podocyte with the recombinant ROBO2 protein of any one of E1-E44,or the pharmaceutical composition of E59.

E64. A method of modulating podocyte function, comprising contactingsaid podocyte with the recombinant ROBO2 protein of any one of E1-E44,or the pharmaceutical composition of E59.

E65. A method of treating glomerular disease, comprising administeringto a subject in need thereof a therapeutically effective amount of therecombinant ROBO2 protein of any one of any one of E1-E44, or thepharmaceutical composition of E59.

E66. A method of treating Focal Segmental Glomerular Sclerosis (FSGS),comprising administering to a subject in need thereof a therapeuticallyeffective amount of the recombinant ROBO2 protein, of any one of E1-E44,or the pharmaceutical composition of E59.

E67. A method of treating nephropathy comprising administering to asubject in need thereof a therapeutically effective amount of therecombinant ROBO2 protein of any one of E1-E44, or the pharmaceuticalcomposition of E59.

E68. The method of E67, wherein said nephropathy is IgA nephropathy.

E69. The method of any one of E60-E68, wherein said subject is a human.

E70. The method of any one of E60-E69, wherein said recombinant ROBO2protein, or pharmaceutical composition is administered intravenously.

E71. The method of any one of E60-E69, wherein said recombinant ROBO2protein, or pharmaceutical composition is administered subcutaneously.

E72. The method of any one of E60-E71, wherein recombinant ROBO2protein, or pharmaceutical composition, is administered about twice aweek, once a week, once every two weeks, once every three weeks, onceevery four weeks, once every five weeks, once every six weeks, onceevery seven weeks, once every eight weeks, once every nine weeks, onceevery ten weeks, twice a month, once a month, once every two months,once every three months, or once every four months.

E73. The recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, for use as a medicament.

E74. The recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, for use in reducing the activity ofROBO2 in a cell.

E75. The recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, for use in reducing the activity ofROBO2 in a subject.

E76. The recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, for use in preserving podocytefunction in a subject.

E77. The recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, for use in modulating podocytefunction in a subject.

E78. The recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, for use in treating a glomerulardisease in a subject.

E79. The recombinant ROBO2 protein of E78, wherein said glomerulardisease is FSGS.

E80. The recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, for use in treating nephropathy in asubject.

E81. The recombinant ROBO2 protein of E80, wherein said nephropathy isan IgA nephropathy.

E82. Use of the recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, in the manufacture of a medicamentfor reducing the activity of ROBO2 in a cell.

E83. Use of the recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, in the manufacture of a medicamentfor reducing the activity of ROBO2 in a subject.

E84. Use of the recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, in the manufacture of a medicamentfor preserving podocyte function in a subject.

E85. Use of the recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, in the manufacture of a medicamentfor modulating podocyte function in a subject.

E86. Use of the recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, in the manufacture of a medicamentfor treating a glomerular disease in a subject.

E87. Use of the recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59, in the manufacture of a medicamentfor treating nephropathy in a subject

E88. A kit comprising a container, a composition within the containercomprising the recombinant ROBO2 protein of any one of E1-E44, or thepharmaceutical composition of E59 and a package insert containinginstructions to administer a therapeutically effective amount of therecombinant ROBO2 protein or the pharmaceutical composition fortreatment of a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graphic presentation showing the domains of human ROBO2.The 21-amino acid ROBO2 leader sequence (SEQ ID NO: 17; residues 1-21according to the numbering set forth in SEQ ID NO: 24) is cleaved duringprotein production to produce mature ROBO2. Residues 22-859 according tothe numbering set forth in SEQ ID NO: 24 form the extracellular domain,residues 860-880 according to the numbering of SEQ ID NO: 24 form thetransmembrane domain, and residues 881-1378 according to the numberingof SEQ ID NO: 24 form the cytoplasmic domain. The ROBO2 pre-Ig1sequence(SEQ ID NO: 8), the Ig1-Ig2 inter-domain linker (SEQ ID NO: 10) and theIg2-Ig3 inter-domain linker (SEQ ID NO: 12) are also shown.

FIG. 1B is a graphic presentation showing exemplary recombinant ROBO2-Fcfusions proteins described herein: ROBO2-Fc. 1.0 (solely contains Ig1domain, i.e., amino acid residues 31 to 127 according to the numberingof SEQ ID NO: 24), ROBO2-Fc. 1.1 (contains Ig1 domain and Ig1-Ig2inter-domain linker, i.e., amino acid residues 31 to 132 according tothe numbering of SEQ ID NO: 24), ROBO2-Fc. 2.0 (contains Ig1 domain,Ig1-Ig2 inter-domain linker, and Ig2 domain, i.e., amino acid residues31 to 220 according to the numbering of SEQ ID NO: 24), ROBO2-Fc. 2.1(contains Ig1 domain, Ig1-Ig2 inter-domain linker, Ig2 domain, andIg2-Ig3 inter-domain linker, i.e., amino acid residues 31 to 224according to the numbering of SEQ ID NO: 24), ROBO2-Fc. 2.2 (containspre-Ig1 sequence, Ig1 domain, Ig1-Ig2 inter-domain linker, Ig2 domain,and Ig2-Ig3 inter-domain linker, i.e., amino acid residues 22 to 224according to the numbering of SEQ ID NO: 24), ROBO2-Fc. 3.0 (containsIg1 domain, Ig1-Ig2 inter-domain linker, Ig2 domain, Ig2-Ig3inter-domain linker, Ig3 domain, i.e., amino acid residues 31 to 309according to the numbering of SEQ ID NO: 24) and ROBO2-Fc. 4.0 (containsIg1 domain, Ig1-Ig2 inter-domain linker, Ig2 domain, Ig2-Ig3inter-domain linker, Ig3 domain, Ig3-Ig4 inter-domain linker and Ig4,i.e., amino acid residues 31 to 409 according to the numbering of SEQ IDNO: 24) described herein.

FIG. 2 shows the ROBO2-Fc 2.2 amino acid sequence (SEQ ID NO: 1).Residues are numbered sequentially starting with the N-terminus. The Ig1and Ig2 domains are shown in all caps, while the Fc domain is shown inlower case. The pre-Ig1 sequence (SEQ ID NO: 8) and the Ig2-Ig3inter-domain linker (SEQ ID NO: 12) are shown in bold and italics, whilethe Ig1-Ig2 inter-domain linker (SEQ ID NO: 10) is shown in italics. Thepredicted intra- and inter-chain disulfide bonds are illustrated withconnecting lines. A single polypeptide chain is shown with disulfidebonds in the Fc hinge region which can dimerize with a second, Fccomprising polypeptide chain. The canonical N-linked glycosylationconsensus sequence sites are circled (i.e., NXS/T where the glycan isattached to the asparagine residue and where X can be any amino acidexcept proline and the third amino acid is either serine or threonine),and the Fc-effector function-null point mutations located at A228, A229,and A231 are shown in bold. The 6-amino acid Gly-Ser linker sequence isshown in the boxed region.

FIG. 3 demonstrates that ROBO2-Fc 2.1 (SEQ ID NO:2) binds to SLIT2 whileROBO2-Fc 1.1 (SEQ ID NO:4) and ROBO2-Fc 2.0 (SEQ ID NO:3) do not.Utilizing the Octet Red ROBO2-Fc proteins were loaded onto antihuman-crystallized fragment (AHFc) sensors at 10 μg/ml and incubatedwith 100 nM SLIT2 for 7 minutes and then the sensors were moved tobuffer alone for 640 seconds. ROBO2-Fc 4.0 (SEQ ID NO: 7) was includedas a positive control for binding. The addition of the sequence VFER(SEQ ID NO: 12) after the Ig2 domain of ROBO2 to create ROBO2-Fc 2.1(SEQ ID NO: 2) was essential to produce a ROBO2-Fc fusion protein thatbinds SLIT2.

FIGS. 4A-4C demonstrate that ROBO2-Fc 2.2 (SEQ ID NO: 1) binds to SLIT2with high affinity. K_(D) values were measured using surface plasmonresonance (SPR). The K_(D) of ROBO2-Fc 2.2 to human/cynomolgus monkeySLIT2-D2 (ROBO2 binding domain, 100% identical) was 0.293 nM (FIG. 4A).The K_(D) of ROBO2-Fc 2.2 to human SLIT2-N(N terminal fragment) was0.279 nM (FIG. 4B), and the K_(D) of ROBO2-Fc 2.2 to rat SLIT2-N was0.543 nM (FIG. 4C).

FIG. 5 demonstrates that ROBO2-Fc 2.2 (SEQ ID NO: 1) binds with highaffinity having an EC₅₀ of 9 nM to human SLIT2-N that is overexpressedon human embryonic kidney (HEK293) cells. A 12-point, 2-fold dilutionseries of ROBO2-Fc 2.2 labeled with Alexa Fluor 647 (AF647) wasincubated with either SLIT2-N expressing HEK293 cells or control HEK293cells. The data are presented as the geometric mean fluorescenceintensity (Geo MFI) of ROBO2-Fc 2.2 AF647 on SLIT2-N HEK293 cells minusthe geometric mean fluorescence intensity of ROBO2-Fc 2.2 AF647 oncontrol HEK293 cells.

FIGS. 6A-6B demonstrate the dose-dependent inhibition of SLIT2-N bindingto cell surface ROBO2 by ROBO2-Fc 2.2 (SEQ ID NO: 1) as assessed byHomogenous Time Resolved Fluorescence (HTRF). An 11-point, 4-fold dosetitration of ROBO-Fc 2.2 (black squares) or an isotype control antibody(black circles) was added to either a human SLIT2-N (FIG. 6A) or ratSLIT2-N (FIG. 6B) human ROBO2 HTRF assay. ROBO2-Fc 2.2 was a potentneutralizer of both human SLIT2-N:human ROBO2 (IC₅₀ of 7 nM) and ratSLIT2-N:human ROBO2 (IC₅₀ of 4 nM) binding.

FIG. 7 depicts the dose-dependent inhibition of SLIT2-N mediatedinhibition of neuronal cell migration by ROBO2-Fc 2.2. Subventricularzone (SVZ) neuronal tissue cell explants were cultured overnight in thepresence of 1 nM SLIT2-N and a dose range of ROBO2-Fc 2.2. ROBO2-Fc 2.2was able to restore neuronal cell migration in a dose-dependent mannerwith an IC₅₀ of 51 nM.

FIG. 8 demonstrates inhibition of proteinuria with treatment of ROBO2-Fc2.2 in the rat Passive Heymann Nephritis model with an exemplaryprophylactic dosing regimen. Twelve animals in each of the indicatedgroups were treated subcutaneously with the indicated dose of ROBO2-Fc2.2 or an irrelevant isotype control monoclonal antibody (control) everythree days starting the day before the induction of the model on day 0.The Y axis indicates the ratio of urine albumin to creatinine (mg/mg) asa measure of leakage of protein into the urine, indicative of podocytedamage. Lewis rats were injected with sheep anti-sera raised against ratkidney brush border (anti-Fx1a, basement membrane and podocytes). Therats developed an immune response to the sheep sera which bound the ratpodocytes. As podocytes are damaged and effaced, proteinuria increases.Treatment with the highest dose of ROBO2-Fc 2.2 at 25 mg/kg reducedproteinuria 45% maximally with a p value less than 0.001 by repeatedmeasure ANOVA statistical analyses compared to the control antibodytreatment. The dose effect was also statistically significant with a pvalue less than 0.001.

FIG. 9 demonstrates inhibition of proteinuria with treatment of ROBO2-Fc2.2 in the rat Passive Heymann Nephritis model with an exemplarytherapeutic dosing regimen. Twelve animals in each of the indicatedgroups were treated subcutaneously with the indicated dose of ROBO2-Fc2.2 or an irrelevant control monoclonal antibody (control) every threedays with the following dosing regimen: control antibody wasadministered on day 0 (circles) and ROBO2-Fc 2.2 was administered on day0 (squares), day 6 (triangles) or day 9 (inverted triangles). The Y axisindicates the ratio of urine albumin to creatinine (mg/mg) as a measureof leakage of protein into the urine, indicative of podocyte damage.Lewis rats were injected with sheep anti-sera raised against rat kidneybrush border (anti-Fx1a, basement membrane and podocytes). The ratsdeveloped an immune response to the sheep sera which bound the ratpodocytes. As podocytes are damaged and effaced, proteinuria increased.Treatment with ROBO2-Fc 2.2 administered on day 0, 6 and 9 reducedproteinuria to a similar extent, 40% maximally and with a p value lessthan 0.001 by repeated measure ANOVA statistical analyses for eachROBO2-Fc 2.2 treated group compared to the control antibody treatment.

FIGS. 10A-10B demonstrate that treatment with ROBO2-Fc 2.2 reducesdamage to podocyte substructure in the Passive Heymann Nephritis Model.Twelve animals in each of the indicated groups were treatedsubcutaneously with the indicated dose of ROBO2-Fc 2.2 or an irrelevantcontrol monoclonal antibody every three days at 25 mg/kg to achieve 100%target coverage starting the day before the induction of the model onday 0. Following animal sacrifice at day 16, selected kidney sampleswere digitally imaged using a transmission electron microscope. Withoutrepetition, three capillary loops of the first three glomeruli found at200× magnification, were imaged at 5000× and 10,000× magnification.ImageJ software (version 1.47v; National Institutes of Health, Bethesda,Md., USA) was used to manually trace and measure the width of adjacentfoot processes as well as the density of slit diaphragms per unit lengthof the glomerular basement membrane (GBM) on high magnificationtransmission electron microscopy images. Samples were analyzed over 3separate experiments. The podocyte foot processes of the ROBO2-Fc 2.2treated animal were significantly shorter than the control antibodytreated animals (FIG. 10A), and the density of slit diaphragms wassignificantly higher in ROBO2-Fc 2.2 treated animals (FIG. 10B; p valueless than 0.01 by two tailed T test) indicating that they were lesseffaced and were protected from the glomerular insult.

FIG. 11 demonstrates that ROBO2-Fc S17T/R73Y binds to human SLIT2-Noverexpressed on human embryonic kidney (HEK293) cells with highaffinity having an EC₅₀ of 2.5 nM. A 12-point, 2-fold dilution series ofROBO2-Fc S17T/R73Y labeled with alexa fluor 647 (AF647) was incubatedwith either SLIT2-N expressing HEK293 cells or control HEK293 cells. Thedata are presented as the geometric mean fluorescence intensity (GeoMFI) of ROBO2-Fc S17T/R73Y AF647 on SLIT2-N HEK293 cells minus thegeometric mean fluorescence intensity of ROBO2-Fc S17T/R73Y AF647 oncontrol HEK293 cells.

FIG. 12 demonstrates the dose-dependent inhibition of SLIT2-N binding tocell surface ROBO2 by ROBO2-Fc S17T/R73Y as assessed by Homogenous TimeResolved Fluorescence (HTRF). An 11-point, 4-fold dose titration ofROBO2-Fc S17T/R73Y (black squares) or an isotype control antibody (blackcircles) was added to a human SLIT2-N human ROBO2 HTRF assay. ROBO2-FcS17T/R73Y was a potent neutralizer of human SLIT2-N:human ROBO2 bindingwith an IC₅₀ of 1.4 nM.

FIG. 13 depicts the dose-dependent inhibition of SLIT2-N mediatedinhibition of neuronal cell migration by ROBO2-Fc S17T/R73Y.Subventricular zone (SVZ) neuronal tissue cell explants were culturedovernight in the presence of 1 nM SLIT2-N and titrated amounts ofROBO2-Fc S17T/R73Y. ROBO2-Fc S17T/R73Y was able to restore neuronal cellmigration in a dose-dependent manner with an IC₅₀ of 11.5 nM.

FIG. 14 shows a drawing depicting the crystal structure of a ROBO2-Hisconstruct that consists of the ROBO2 pre-Ig1 sequence (SRLRQEDFP; SEQ IDNO: 8), Ig1 domain, Ig2 domain and the ROBO2 Ig2-3 inter-domain linker(VFER; SEQ ID NO: 12) with a 6× histidine tag (His6) (SEQ ID NO: 25) atthe C-terminus. The crystal structure of ROBO2-His reveals that thatAsp7, Phe8, and Pro9 are substantially involved in the interactionsvital for structural integrity of ROBO2's Ig1 domain.

FIG. 15 shows a drawing depicting the crystal structure of the ROBO2-Hisconstruct. The crystal structure of ROBO2-His6 (“His6” disclosed as SEQID NO: 25) reveals that the ROBO2 Ig2-3 inter-domain linker,Valine200-Phenylalanine201-Glutamic acid202-Arginine203 (SEQ ID NO: 12),effectively stabilizes the structural fold in the C-terminal region ofROBO2's Ig2 domain.

FIG. 16 shows the crystal structure of the first immunoglobulin-likedomain (Ig1) of ROBO2 S17T/R73Y in complex with SLIT2.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

The invention encompasses novel recombinant ROBO2 proteins capable ofbinding SLIT ligands (for example, the SLIT2 ligand), thereby inhibitingthe interaction of SLIT with ROBO2, and consequently, inhibiting theSLIT2-ROBO2 signaling pathway. Previous studies have shown that whileboth immunoglobulin-like (Ig-like) domains 1 and 2 (Ig1 and Ig2) ofROBO2 interact with SLIT ligands; the first Ig-like domain (Ig1) is theprimary binding site for SLIT. In addition, previous studies haveindicated that removing the three fibronectin type III (FNIII) repeatshas a greater negative effect on ROBO binding to SLIT ligands thanremoval of the third and fourth immunoglobulin-like domains (Ig3 andIg4). That is, FNIII deletion causes a greater reduction in ROBO bindingto SLIT ligands than deletion of Ig3 and Ig4.

Surprisingly, it is now shown for the first time that a construct,ROBO2-Fc 2.1 (SEQ ID NO: 2; FIG. 1B), comprising only the first twoimmunoglobulin-like domains (Ig1 and Ig2) along with the Ig2-3inter-domain linker, VFER (SEQ ID NO: 12), and devoid of the threefibronectin type III (FNIII) repeats bound SLIT2 (FIG. 3). In contrast,recombinant ROBO2 proteins lacking the three fibronectin type III(FNIII) repeats but consisting of:

-   -   (i) the Ig1 domain of ROBO2 (ROBO2-Fc 1.1; SEQ ID NO: 4; FIG.        1B),    -   (ii) the Ig1 and Ig2 domains of ROBO2 (ROBO2-Fc 2.0; SEQ ID NO:        3; FIG. 1B), or    -   (iii) the Ig1, Ig2 and Ig3 domains of ROBO2 (ROBO2-Fc 3.0; SEQ        ID NO: 6; FIG. 1B) did not bind SLIT2 (FIG. 3).

Thus, addition of VFER (SEQ ID NO: 12) to the C-terminus of the Ig1-Ig2domains was required to create a ROBO2-Fc construct (ROBO2-Fc 2.1; SEQID NO: 2) with a robust binding profile to SLIT2 in the absence of theFNIII repeats. This ROBO2-Fc 2.1 construct lacks not only the third,fourth and fifth immunoglobulin-like domains (Ig3, Ig4 and Ig5), but isalso devoid of the three fibronectin type III (FNIII) repeats.Surprisingly, the difference in binding or not binding SLIT2 was foundto be the presence of the four-amino acid VFER sequence (SEQ ID NO: 12).

None of the recombinant ROBO2 protein constructs described above containthe ROBO2 pre-Ig1 sequence (SEQ ID NO: 8). It was discovered,surprisingly, that the production of these recombinant ROBO2 proteins,disclosed and exemplified herein, can be dramatically increased byincluding the ROBO2 pre-Ig1 sequence (SEQ ID NO: 8). Addition of thissequence increased protein production by about 25-fold compared toconstructs lacking the sequence while preserving high affinity bindingto SLIT2 (FIGS. 3-4A-C). As shown in FIG. 14, this ROBO2 pre-Ig1sequence bridges together the two β-sheets of ROBO2's Ig1 domain and isbelieved to stabilize the structural fold of the N-terminal region.Without wishing to be bound by any particular theory, the pre-Ig1sequence appears to contribute to the enhanced expression of the novelproteins.

2. Definitions

In some aspects, provided herein are recombinant ROBO2 proteins capableof binding SLIT and comprising an immunoglobulin domain.

The term “recombinant protein” refers to a polypeptide which is producedby recombinant DNA techniques, wherein generally, DNA encoding thepolypeptide is inserted into a suitable expression vector which, inturn, is introduced into a host cell to produce the recombinant protein.As used herein, “protein” refers to any composition comprising aminoacids and recognized as a protein by those of skill in the art. Theterms “protein”, “peptide” and “polypeptide are used interchangeablyherein. Amino acids may be referred to by their complete names (e.g.,alanine) or by the accepted one letter (e.g., A), or three letter (e.g.,Ala) abbreviations.

As used herein, an “immunoglobulin domain” is a polypeptide derived froman immunoglobulin. In some embodiments, an immunoglobulin domaincomprises an immunoglobulin heavy chain or a portion thereof. In someembodiments, the portion of the heavy chain is the crystallizablefragment (Fc) or a portion thereof. As used herein, the Fc fragmentcomprises the heavy chain hinge region, and the C_(H)2 and C_(H)3domains of the heavy chain of an immunoglobulin. The heavy chain (orportion thereof) may be derived from any one of the known heavy chainisotypes: IgG (γ), IgM (μ), IgD (δ), IgE (ε), or IgA (α). In addition,the heavy chain (or portion thereof) may be derived from any one of theknown heavy chain isotypes or subtypes: IgG1 (γ1), IgG2 (γ2), IgG3 (γ3),IgG4 (γ4), IgA1 (α1), IgA2 (α2). In some embodiments, the immunoglobulindomain comprises an uninterrupted native (i.e., wild-type) sequence ofan immunoglobulin. In some embodiments, the immunoglobulin Fc domaincomprises a variant Fc region.

For all heavy chain constant region amino acid positions discussed inthe present invention, numbering is according to the Eu index firstdescribed in Edelman et al., 1969, Proc. Natl. Acad. Sci. USA63(1):78-85, describing the amino acid sequence of myeloma protein Eu,which is the first human IgG1 sequenced. The Eu index of Edelman et al.is also set forth in Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda. Thus, the “EU index as setforth in Kabat” or “EU index of Kabat” refers to the residue numberingsystem based on the human IgG1 Eu antibody of Edelman et al. as setforth in Kabat 1991.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” comprises an amino acid sequence which differs from that of anative sequence Fc region by virtue of at least one amino acidmodification. Preferably, the variant Fc region has at least one aminoacid substitution compared to a native sequence Fc region, e.g., fromabout one to about ten amino acid substitutions, and preferably, fromabout one to about five amino acid substitutions compared to a nativesequence Fc region. The variant Fc region herein will preferably possessat least about 80% amino acid sequence identity with a native sequenceFc region, and more preferably, at least about 90% amino acid sequenceidentity therewith, more preferably, at least about 95%, at least about96%, at least about 97%, at least about 98%, and most preferably atleast about 99% amino acid sequence identity therewith.

As used herein a “linker” is a molecule or group of molecules that bindstwo separate entities (e.g., the extracellular domain and theimmunoglobulin domain of a recombinant ROBO2-Fc protein) to one anotherand can provide spacing and flexibility between the two entities suchthat they are able to achieve a conformation in which they, e.g.,specifically bind their cognate ligand (e.g., SLIT ligand). Proteinlinkers are particularly preferred, and they may be expressed as acomponent of the recombinant protein using standard recombinant DNAtechniques well-known in the art.

The term “IC₅₀” or “the half maximal inhibitory concentration” refers tothe concentration of the recombinant ROBO2 protein that is required for50% inhibition of the ROBO2-SLIT signaling pathway, for example theROBO2-SLIT2 signaling pathway. IC₅₀ is a measure of how much ofrecombinant ROBO2 protein is needed to inhibit a ROBO2-SLIT biologicalprocess by 50%, such as the binding between ROBO2 and a SLIT ligand,binding of intracellular signaling molecules (such as srGAP1 or Nck) tothe intracellular domain of ROBO2 and/or downstream activities ofROBO2-SLIT signaling (such as actin polymerization, podocyte adhesion,and/or SLITx-N mediated inhibition of neuronal cell migration). A lowerIC₅₀ indicates a more potent effect since a smaller amount of therecombinant ROBO2 protein mediates a more potent inhibitory effect.

As used herein, the term “SLITx” refers generally to a SLIT ligand.Similarly, the terms “SLITx-N” and SLITx-C″ refer generally toN-terminal and C-terminal fragments, respectively, of SLIT ligands. TheSLIT ligand may be a mammalian SLIT ligand, preferably a human SLITligand. In some embodiments, the SLIT ligand is selected from the groupconsisting of a SLIT1 ligand, a SLIT2 ligand, and a SLIT3 ligand. TheSLIT ligand may be a SLIT2 ligand, preferably a human SLIT2 ligand.

As used herein, a “subject” is an animal, preferably a mammal, morepreferably a non-human primate, and most preferably a human. The terms“subject” “individual” and “patient” are used interchangeably herein. Inall embodiments, human nucleic acids and human polypeptides arepreferred. It is believed that the results obtained using the human, ratand cynomolgus monkey molecules described elsewhere herein arepredictive of the results that may be obtained using other homologoussequences.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: reducing proteinuria (i.e., reducing the amount ofprotein in the urine compared with the level of protein in urine in theabsence of drug administration), reducing edema, and/or restoring bloodalbumin levels. The term “treatment” includes prophylactic and/ortherapeutic treatments. If it is administered prior to clinicalmanifestation of a condition, the treatment is considered prophylactic.Therapeutic treatment includes, e.g., ameliorating or reducing theseverity of a disease, or shortening the length of the disease.

The term “therapeutically effective amount” refers to an amount of atherapeutic agent of this invention effective to “treat” a disease ordisorder in a subject. For example, a therapeutically effective amountmay be the amount that alleviates one or more symptoms of the disease orthe amount necessary to keep a disease in remission. In the case of afocal segmental glomerulosclerosis (FSGS), the therapeutically effectiveamount refers to that amount which has at least one of the followingeffects: reducing proteinuria (i.e., reducing the amount of protein inthe urine compared with the level of protein in urine in the absence ofdrug administration), reducing edema, and/or restoring blood albuminlevels.

“About” or “approximately” when used in connection with a measurablenumerical variable, refers to the indicated value of the variable and toall values of the variable that are within the experimental error of theindicated value (e.g., within the 95% confidence interval for the mean)or ±10% of the indicated value, whichever is greater. Numeric ranges areinclusive of the numbers defining the range.

Binding Affinity

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a contact residue of one bindingpartner (e.g., the recombinant ROBO2 protein disclosed herein) and acontact residue of its binding partner (e.g., a SLIT ligand). Unlessindicated otherwise, as used herein, “binding affinity” refers tobinding affinity that reflects a 1:1 interaction between members of abinding pair or binding partners (e.g., the recombinant ROBO2 proteinand a SLIT2 ligand).

At its most detailed level, the binding affinity for the interactionbetween ROBO2 and a SLIT ligand can be defined by the spatialcoordinates defining the atomic contacts present in the ROBO2/SLITinteraction, as well as information about their relative contributionsto the binding thermodynamics. At one level, a contact residue can becharacterized by the spatial coordinates defining the atomic contactsbetween ROBO2 and SLIT. In one aspect, the contact residue can bedefined by a specific criterion, e.g., distance between atoms in theROBO2 protein amino acid residue and the atoms in the SLIT protein aminoacid residue (e.g., a distance of equal to or less than about 4 Å (suchas 3.8 Å used in the Examples here) from a heavy atom of a ROBO2 aminoacid residue and a heavy atom of an amino acid residue of SLIT. Inanother aspect, a contact residue can be characterized as participatingin a hydrogen bond interaction with the cognate binding partner, or witha water molecule that is also hydrogen bonded to the binding partner(i.e., water-mediated hydrogen bonding). In another aspect, a contactresidue can be characterized as forming a salt bridge with a residue ofthe binding partner. In yet another aspect, a contact residue can becharacterized as a residue having a non-zero change in buried surfacearea (BSA) due to interaction with a contact residue of the bindingpartner. At a less detailed level, the binding affinity can becharacterized through function, e.g., by competition binding with otherproteins.

Low-affinity recombinant proteins generally bind their ligands slowlyand tend to dissociate readily, whereas high-affinity recombinantproteins generally bind their ligands faster and tend to remain boundlonger. A variety of methods of measuring binding affinity are known inthe art, any of which can be used for purposes of the present invention.Specific illustrative and exemplary embodiments for measuring bindingaffinity are described in the following.

The binding affinity can be expressed as K_(D) value, which refers tothe dissociation rate of a particular recombinant ROBO2 protein-SLITligand interaction. K_(D) is the ratio of the rate of dissociation, alsocalled the “off-rate (k_(off))”, to the association rate, or “on-rate(k_(on))”. Thus, K_(D) equals k_(off)/k_(on) and is expressed as a molarconcentration (M), and the smaller the K_(D), the stronger the affinityof binding. K_(D) values can be determined using methods wellestablished in the art. One exemplary method for measuring K_(D) issurface plasmon resonance (SPR), a method well-known in the art (e.g.,Nguyen et al. Sensors (Basel). 2015 May 5; 15(5):10481-510). K_(D) valuemay be measured by SPR using a biosensor system such as a BIACORE®system. BIAcore kinetic analysis comprises analyzing the binding anddissociation of an antigen from chips with immobilized molecules (e.g.molecules comprising epitope binding domains), on their surface. Anotherwell-known method in the art for determining the K_(D) of a protein isby using Bio-Layer Interferometry (e.g., Shah et al. J Vis Exp. 2014;(84): 51383). K_(D) value may be measured using OCTET® technology (OctetQKe system, ForteBio). Alternatively or in addition, a KinExA® (KineticExclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.)can also be used. Any method known in the art for assessing the bindingaffinity between two binding partners is encompassed herein.

3. Recombinant ROBO2 Proteins

In some aspects, the instant disclosure provides recombinant ROBO2proteins. In some embodiments, the recombinant ROBO2 proteins disclosedherein bind SLIT ligands (in particular, SLIT2 ligand), therebypreventing the binding of SLIT to cellular ROBO2 receptors, and arehence referred to as SLIT neutralizing ligand traps. Surprisingly, asshown in the Examples, a construct, ROBO2-Fc 2.1 (SEQ ID NO: 2; FIG.1B), comprising the first two immunoglobulin-like domains (Ig1 and Ig2),Ig1-Ig2 inter-domain linker, along with the Ig2-3 inter-domain linker,VFER (SEQ ID NO: 12), and devoid of the three fibronectin type III(FNIII) repeats bound SLIT2 (FIG. 3). Crystal structure studies alsoshow that the inclusion of the ROBO2 Ig2-Ig3 inter-domain linker, VFER(V200-F201-E202-R203; SEQ ID NO: 12) effectively stabilizes thestructural fold in the C-terminal region of ROBO2's second Ig domainFIG. 15), and notably increases the expression level of the recombinantROBO2 protein.

In some aspects, the instant disclosure provides recombinantpolypeptides comprising a SLIT-binding moiety and a half-life extendingmoiety. The “SLIT-binding moiety” confers SLIT-binding ability to therecombinant ROBO2 protein. In some embodiments, the SLIT-binding moietycomprises a portion of a ROBO2 extracellular domain. In someembodiments, the portion of the ROBO2 extracellular domain comprises atleast two immunoglobulin-like (Ig-like) domains, and a C-terminussequence consisting of VFER (SEQ ID NO: 12). In some embodiments, the atleast two Ig-like domains are selected from the group consisting of Ig1,Ig2, Ig3, Ig4 and Ig5. In some embodiments, the at least two Ig-likedomains are selected from the group consisting of the sequence of SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14. In someembodiments, the portion of the ROBO2 extracellular domain comprises thefirst two Ig-like domains (Ig1 and Ig2) of ROBO2, In some embodiments,the portion of the ROBO2 extracellular domain comprises SEQ ID NO: 9and/or SEQ ID NO: 11.

Protein production studies also determined that the production of therecombinant ROBO2 proteins, disclosed and exemplified herein, can bedramatically increased by including the ROBO2 pre-Ig1 sequence (SEQ IDNO: 8). Addition of this sequence increases protein production intransiently and/or stably transfected mammalian cells by about 25-foldwhile preserving high affinity binding to SLIT (FIGS. 3-5).

Accordingly, in some embodiments, the portion of the ROBO2 extracellulardomain further comprises the ROBO2 pre-Ig1 sequence. In someembodiments, the ROBO2 pre-Ig1 sequence comprises SEQ ID NO: 8.

In some embodiments, the portion of the ROBO2 extracellular domaincomprises ROBO2 pre-Ig1 sequence, Ig1, Ig1-Ig2 inter-domain linker, Ig2,and Ig2-Ig3 inter-domain linker. Exemplary sequences of the ROBO2pre-Ig1 sequence (SEQ ID NO: 8), Ig1 (SEQ ID NO: 9), Ig1-Ig2inter-domain linker (SEQ ID NO: 10), Ig2 (SEQ ID NO: 11), and Ig2-Ig3inter-domain linker (SEQ ID NO: 12) are shown in Table 23 and alsoillustrated in FIG. 2. The present invention is not limited to thesequences disclosed herein. Corresponding residues from other ROBO2homologs, isoforms, variants, or fragments can be identified accordingto sequence alignment or structural alignment that is known in the art.For example, alignments can be done by hand or by using well-knownsequence alignment programs such as ClustalW2, or “BLAST 2 Sequences”using default parameters.

In some embodiments, the portion of the ROBO2 extracellular domaincomprises amino acid residues 1 to 203 according to the numbering of SEQID NO: 1. In some embodiments, the recombinant ROBO2 protein comprises aportion of the ROBO2 extracellular domain that shares at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to aminoacid residues 1 to 203 according to the numbering of SEQ ID NO: 1. Insome embodiments, the recombinant ROBO2 protein comprises anextracellular domain consisting of amino acid residues 1 to 203according to the numbering of SEQ ID NO: 1.

In some aspects, the ROBO2 is human ROBO2. In some aspects, the ROBO2 israt ROBO2. In some aspects, the ROBO2 is mouse ROBO2. In some aspects,the ROBO2 is primate ROBO2. In some aspects, the ROBO2 is ape ROBO2. Insome aspects, the ROBO2 is monkey ROBO2. In some aspects, the ROBO2 iscynomologus monkey ROBO2.

In addition to the SLIT-binding moiety, the novel, recombinant ROBO2proteins comprise a half-life extending moiety. The “half-life extendingmoiety” extends the serum half-life in vivo of the recombinant ROBO2protein compared to the same ROBO2 protein without the half-lifeextending moiety. Examples of half-life extending moieties include, butare not limited to, polyhistidine, Glu-Glu, glutathione S transferase(GST), thioredoxin, protein A, protein G, an immunoglobulin domain,maltose binding protein (MBP), human serum albumin (HSA), orpolyethylene glycol (PEG). In some embodiments, the half-life extendingmoiety comprises an immunoglobulin domain. In some embodiments, theimmunoglobulin domain comprises an Fc domain. In some embodiments, theFc domain is derived from any one of the known heavy chain isotypes: IgG(γ), IgM (μ), IgD (δ), IgE (ε), or IgA (α). In some embodiments, the Fcdomain is derived from any one of the known heavy chain isotypes orsubtypes: IgG₁ (γ1), IgG₂ (γ2), IgG₃ (γ3), IgG₄ (γ4), IgA₁ (α1), IgA₂(α2). In some embodiments, the Fc domain is the Fc domain of human IgG₁.

In some embodiments, the Fc domain comprises an uninterrupted nativesequence (i.e., wild type sequence) of a Fc domain. In some embodiments,the immunoglobulin Fc domain comprises a variant Fc domain resulting inaltered biological activity. For example, at least one point mutation ordeletion may be introduced into the Fc domain so as to reduce oreliminate the effector activity (e.g., WO 2005/063815), and/or toincrease the homogeneity during the production of the recombinantprotein. In some embodiments, the Fc domain is the Fc domain of humanIgG₁ and comprises one or more of the following effector-nullsubstitutions: L234A, L235A, and G237A (Eu numbering) or L228A, L229Aand G231A relative to the numbering of SEQ ID NO: 1. In someembodiments, the Fc domain does not comprise the lysine located at theC-terminal position of human IgG1 (i.e., K447 by Eu numbering). Theabsence of the lysine may increase homogeneity during the production ofthe recombinant protein. In some embodiments, the Fc domain comprisesthe lysine located at the C-terminal position (K447, Eu numbering).

In some embodiments, the recombinant ROBO2 polypeptide comprises one,two, three or four intra-chain disulfide bonds which may be located inthe ROBO2 extracellular domain or in the Fc domain. In some embodiments,the recombinant ROBO2 polypeptide comprises four intra-chain disulfidebonds, two of which are located in the ROBO2 extracellular domain andtwo are located in the Fc domain. In some embodiments, the intra-chaindisulfide bonds located in the ROBO2 extracellular domain are betweenCys31 and Cys89, and between Cys133 and Cys182, all according to thenumbering of SEQ ID NO: 1. In some embodiments, the intra-chaindisulfide bonds located in the Fc domain are between Cys255 and Cys315,and between Cys361 and Cys419 all according to the numbering of SEQ IDNO: 1.

In some embodiments, two of the recombinant ROBO2 polypeptidesassociate, either covalently, for example, by a disulfide bond, apolypeptide bond or a crosslinking agent, or non-covalently, to producea homodimeric protein. In some embodiments, two recombinant ROBO2polypeptides are associated covalently to form a homodimer by means ofat least one, and more preferably, two inter-chain disulfide bonds viacysteine residues, preferably located within the immunoglobulin Fcregion of each polypeptide. In some embodiments, the two inter-chaindisulfide bonds are between Cys220 and Cys223. In some embodiments, lessthan 90%, less than 80%, less than 70%, less than 60%, less than 50%,less than 40%, less than 30%, less than 20%, less than 10%, less than8%, less than 5%, less than 4%, less than 2%, less than 1% of therecombinant ROBO2 polypeptides are associated to form a homodimer.

In some embodiments, a recombinant ROBO2 polypeptide associates withanother polypeptide, either covalently, for example, by a disulfidebond, a polypeptide bond or a crosslinking agent, or non-covalently, toproduce a heterodimeric protein. In some embodiments, the heterodimericprotein is bispecific or multispecific. In some embodiments the otherpolypeptide comprises an immunoglobulin domain. In some embodiments, thepolypeptides are associated covalently to form a heterodimer by means ofat least one, and more preferably, two inter-chain disulfide bonds viacysteine residues, preferably located within the immunoglobulin Fcregion of each polypeptide. In some embodiments, the two inter-chaindisulfide bonds are between Cys220 and Cys223 of the recombinant ROBO2polypeptide. In some embodiments, the heterodimeric protein comprisestwo different recombinant ROBO2 polypeptides.

In some embodiments, the Fc domain of the recombinant ROBO2 proteincomprises amino acid residues 210 to 440 according to the numbering ofSEQ ID NO: 1. In some embodiments, the recombinant ROBO2 proteincomprises a Fc domain sharing at least 90%, at least 91%, at least 92%,at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% amino acid sequence identity to amino acidresidues 210 to 440 according to the numbering of SEQ ID NO: 1. In someembodiments, the recombinant ROBO2 protein comprises a Fc domainconsisting of amino acid residues 210 to 440 of according to thenumbering SEQ ID NO: 1.

In some embodiments, the extracellular domain of the recombinant ROBO2protein is contiguous with the immunoglobulin domain. That is, the lastC-terminal amino acid residue of the extracellular domain of the ROBO2protein is covalently linked by a peptidyl bond with the firstN-terminal amino acid residue of the immunoglobulin domain. In someembodiments, the extracellular domain of the recombinant ROBO2 proteinis connected via a linker to the immunoglobulin domain. In someembodiments, the linker is a peptidyl linker. In some embodiments, thepeptidyl linker comprises about 1 to 30 amino acid residues. In someembodiments, the peptidyl linker is selected from the group consistingof a glycine rich peptide; a peptide comprising glycine and serine; apeptide having a sequence [Gly-Gly-Ser]_(n), wherein n is 1, 2, 3, 4, 5,or 6 (SEQ ID NO: 22); and a peptide having a sequence[Gly-Gly-Gly-Gly-Ser]_(n), wherein n is 1, 2, 3, 4, 5, or 6 (SEQ ID NO:23). In some embodiments, the peptidyl linker is Gly-Gly-Ser-Gly-Gly-Ser(SEQ ID NO: 15). A glycine rich peptide linker comprises a peptidelinker, wherein at least 25% of the residues are glycine. Glycine richpeptide linkers are well known in the art (e.g., Chichili et al. ProteinSci. 2013 February; 22(2): 153-167).

In some embodiments, the recombinant ROBO2-Fc protein comprises thesequence of SEQ ID NO: 1. In some embodiments, the recombinant ROBO2-Fcprotein consists of the sequence of SEQ ID NO: 1. In some embodiments,the recombinant ROBO2-Fc protein comprises an amino acid sequence havingat least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identity to the sequence of SEQ ID NO: 1. In some embodiments, therecombinant ROBO2-Fc protein comprises an amino acid sequence having atleast 95% identity to the sequence of SEQ ID NO: 1. In some embodiments,the recombinant ROBO2-Fc protein comprises an amino acid sequence havingat least 96% identity to the sequence of SEQ ID NO: 1. In someembodiments, the recombinant ROBO2-Fc protein comprises an amino acidsequence having at least 97% identity to the sequence of SEQ ID NO: 1.In some embodiments, the recombinant ROBO2-Fc protein comprises an aminoacid sequence having at least 98% identity to the sequence of SEQ IDNO: 1. In some embodiments, the recombinant ROBO2-Fc protein comprisesan amino acid sequence having at least 99% identity to the sequence ofSEQ ID NO: 1.

In some embodiments, no more than 10, no more than 9, no more than 8, nomore than 7, no more than 6, no more than 5, no more than 4, no morethan 3, no more than 2, or no more than 1 substitution is made relativeto the sequence of SEQ ID NO: 1. In some embodiments, no more than 5substitutions are made relative to the sequence of SEQ ID NO: 1. In someembodiments, no more than 4 substitutions are made relative to thesequence of SEQ ID NO: 1. In some embodiments, no more than 3substitutions are made relative to the sequence of SEQ ID NO: 1. In someembodiments, no more than 2 substitutions are made relative to thesequence of SEQ ID NO: 1. In some embodiments, no more than 1substitution is made relative to the sequence of SEQ ID NO: 1. In someembodiments, the substitution(s) do not change the K_(D) by more than1000-fold, more than 100-fold, or 10-fold compared to the K_(D) of theprotein comprising the sequence of SEQ ID NO: 1. In certain embodiments,the substitution is a conservative substitution according to Table 1.

TABLE 1 Amino Acid Substitutions Original Conservative Exemplary ResidueSubstitutions Substitutions Ala (A) Val Val; Leu; Ile Arg (R) Lys Lys;Gln; Asn Asn (N) Gln Gln; His; Asp, Lys; Arg Asp (D) Glu Glu; Asn Cys(C) Ser Ser; Ala Gln (Q) Asn Asn; Glu Glu (E) Asp Asp; Gln Gly (G) AlaAla His (H) Arg Asn; Gln; Lys; Arg Ile (I) Leu Leu; Val; Met; Ala; Phe;Norleucine Leu (L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys (K) ArgArg; Gln; Asn Met (M) Leu Leu; Phe; Ile Phe (F) Tyr Leu; Val; Ile; Ala;Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr; PheTyr(Y) Phe Trp; Phe; Thr; Ser Val (V) Leu Ile; Leu; Met; Phe; Ala;Norleucine

In some embodiments, one of more of the ROBO2 amino acid residues listedin Tables 4-15 are not substituted (for example, E6, D7, F8, P9, V200,F201, E202, R203 each numbered relative to SEQ ID NO: 1). In someembodiments, none of the amino acid resides listed in Tables 4-15 (forexample, E6, D7, F8, P9, V200, F201, E202, R203 numbered relative to SEQID NO: 1) are substituted. ROBO2 amino acid residues disclosed in Tables4-15 are amino acid residues believed to be important for supporting thestructural integrity of the SLIT-binding domain, according to thecrystal structure study (see Example 5). Amino acid substitutions atthese positions could potentially affect SLIT binding. Accordingly, itmay be desirable that the substitution does not occur at thesepositions. In some embodiments, the recombinant ROBO2 protein comprisesa portion of the ROBO2 extracellular domain that shares at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identity to theamino acid residues 1 to 203 of the sequence set forth in SEQ ID NO: 1,and further comprises one or more residues E6, D7, F8, P9, V200, F201,E202, and R203 (numbering according to the sequence of SEQ ID NO:1).

In some aspects, one or more point mutations relative to the sequence ofSEQ ID NO: 1 may be introduced to increase the affinity of therecombinant ROBO2 protein to a SLIT ligand, e.g., SLIT2. As shown in theExamples, the binding affinity to SLIT2 can be increased by about10-fold by introducing point mutations S17T and R73Y relative to thesequence of SEQ ID NO: 1 (FIGS. 11-13). Accordingly, in some aspects,the instant disclosure provides a recombinant ROBO2 protein having oneor more of the following mutations: S17T and R73Y relative to thesequence of SEQ ID NO: 1. In some embodiments, the recombinant ROBO2protein comprises the sequence set forth in SEQ ID NO: 19. In someembodiments, the recombinant ROBO2 protein consists of the sequence setforth SEQ ID NO: 19. In some embodiments, the recombinant ROBO2 proteindoes not comprise the C-terminal lysine located at amino acid residue441 according to the numbering of SEQ ID NO: 19. In certain embodiments,the recombinant ROBO2 proteins of the invention have a K_(D) of not morethan about 1×10⁻⁶ M, such as not more than about 1×10⁻⁷ M, not more thanabout 9×10⁻⁸ M, not more than about 8×10⁻⁸ M, not more than about 7×10⁻⁸M, not more than about 6×10⁻⁸ M, not more than about 5×10⁻⁸ M, not morethan about 4×10⁻⁸ M, not more than about 3×10⁻⁸ M, not more than about2×10⁻⁸ M, not more than about 1×10⁻⁸ M, not more than about 9×10⁻⁹ M,not more than about 8×10⁻⁹ M, not more than about 7×10⁻⁹ M, not morethan about 6×10⁻⁹ M, not more than about 5×10⁻⁹ M, not more than about4×10⁻⁹ M, not more than about 3×10⁻⁹ M, not more than about 2×10⁻⁹ M,not more than about 1×10⁻⁹ M, not more than about 9×10⁻¹⁰ M, not morethan about 8×10⁻¹⁰ M, not more than about 7×10⁻¹⁰ M, not more than about6×10⁻¹⁰ M, not more than about 5×10⁻¹⁰ M, not more than about 4×10⁻¹⁰ M,not more than about 3×10⁻¹⁰ M, not more than about 2×10⁻¹⁰ M, not morethan about 1×10⁻¹⁰ M, not more than about 9×10⁻¹¹ M, not more than about8×10⁻¹¹ M, not more than about 7×10⁻¹¹ M, not more than about 6×10⁻¹¹ M,not more than about 5×10⁻¹¹ M, not more than about 4×10⁻¹¹ M, not morethan about 3×10⁻¹¹ M, not more than about 2×10⁻¹¹ M, not more than about1×10⁻¹¹ M, not more than about 9×10⁻¹² M, not more than about 8×10⁻¹² M,not more than about 7×10⁻¹² M, not more than about 6×10⁻¹² M, not morethan about 5×10⁻¹² M, not more than about 4×10⁻¹² M, not more than about3×10⁻¹² M, not more than about 2×10⁻¹² M, not more than about 1×10⁻¹² M,not more than about 9×10⁻¹³ M, not more than about 8×10⁻¹³ M, not morethan about 7×10⁻¹³ M, not more than about 6×10⁻¹³ M, not more than about5×10⁻¹³ M, not more than about 4×10⁻¹³ M, not more than about 3×10⁻¹³ M,not more than about 2×10⁻¹³ M, not more than about 1×10⁻¹³ M.

In certain embodiments, the recombinant ROBO2 proteins of the inventionhave a K_(D) ranging from about 1×10⁻⁷M to about 1×10⁻¹⁴ M, from about9×10⁻⁸M to about 1×10⁻¹⁴ M, from about 8×10⁻⁸M to about 1×10⁻¹⁴ M, fromabout 7×10⁻⁸M to about 1×10⁻¹⁴ M, from about 6×10⁻⁸M to about 1×10⁻¹⁴ M,from about 5×10⁻⁸M to about 1×10⁻¹⁴ M, from about 4×10⁻⁸M to about1×10⁻¹⁴ M, from about 3×10⁻⁸ M to about 1×10⁻¹⁴ M, from about 2×10⁻⁸ Mto about 1×10⁻¹⁴ M, from about 1×10⁻⁸M to about 1×10⁻¹⁴ M, from about9×10⁻⁹M to about 1×10⁻¹⁴ M, from about 8×10⁻⁹M to about 1×10⁻¹⁴ M, fromabout 7×10⁻⁹M to about 1×10⁻¹⁴ M, from about 6×10⁻⁹M to about 1×10⁻¹⁴ M,from about 5×10⁻⁹M to about 1×10⁻¹⁴ M, from about 4×10⁻⁹M to about1×10⁻¹⁴ M, from about 3×10⁻⁹M to about 1×10⁻¹⁴ M, from about 2×10⁻⁹M toabout 1×10⁻¹⁴ M, from about 1×10⁻⁹M to about 1×10⁻¹⁴ M, from about1×10⁻⁷M to about 1×10⁻¹³ M, from about 9×10⁻⁸M to about 1×10⁻¹³ M, fromabout 8×10⁻⁸M to about 1×10⁻¹³ M, from about 7×10⁻⁸M to about 1×10⁻¹³ M,from about 6×10⁻⁸M to about 1×10⁻¹³ M, from about 5×10⁻⁸M to about1×10⁻¹³ M, from about 4×10⁻⁸ M to about 1×10⁻¹³ M, from about 3×10⁻⁸ Mto about 1×10⁻¹³ M, from about 2×10⁻⁸ M to about 1×10⁻¹³ M, from about1×10⁻⁸M to about 1×10⁻¹³ M, from about 9×10⁻⁹M to about 1×10⁻¹³ M, fromabout 8×10⁻⁹M to about 1×10⁻¹³ M, from about 7×10⁻⁹M to about 1×10⁻¹³ M,from about 6×10⁻⁹M to about 1×10⁻¹³ M, from about 5×10⁻⁹M to about1×10⁻¹³ M, from about 4×10⁻⁹M to about 1×10⁻¹³ M, from about 3×10⁻⁹M toabout 1×10⁻¹³ M, from about 2×10⁻⁹M to about 1×10⁻¹³ M, or from about1×10⁻⁹M to about 1×10⁻¹³ M.

In some embodiments, the recombinant ROBO2 protein binds SLIT2 with aK_(D) of or less than: about 10 nM, about 5 nM, about 2 nM, about 1 nM,about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM,about 400 pM, about 300 pM, about 250 pM, about 200 pM, about 150 pM,about 100 pM, about 50 pM, about 40 pM, about 30 pM, about 25 pM, about20 pM, about 15 pM, about 10 pM, about 5 pM, or about 1 pM. In someembodiments, the recombinant ROBO2 protein binds SLIT2 with a K_(D) ofabout 600 pM. In some embodiments, the recombinant ROBO2 protein bindsSLIT2 with a K_(D) of about 500 pM. In some embodiments, the recombinantROBO2 protein binds SLIT2 with a K_(D) of about 400 pM. In someembodiments, the recombinant ROBO2 protein binds SLIT2 with a K_(D) ofabout 300 pM. In some embodiments, the recombinant ROBO2 protein bindsSLIT2 with a K_(D) of about 250 pM. In some embodiments, the recombinantROBO2 protein binds SLIT2 with a K_(D) of about 200 pM.

In general, a recombinant ROBO2-Fc protein should bind to a SLIT ligand(e.g., SLIT2) with high affinity, in order to effectively block theactivities of ROBO2. It is desirable that the recombinant ROBO2-Fcprotein have binding affinities (K_(D)) in low nanomolar and picomolarrange, such as about 1×10⁻⁸ M or lower.

In some embodiments, the recombinant ROBO-Fc protein binds SLIT2 with aK_(D) that is at least about 2-fold, about 4-fold, about 6-fold, about8-fold, about 10-fold, about 20-fold, about 40-fold, about 60-fold,about 80-fold, about 100-fold, about 120-fold, about 140-fold, about160-fold, lower than the K_(D) value for binding of ROBO1 to SLIT2. Insome embodiments, the recombinant ROBO2-Fc protein binds SLIT2 with aK_(D) that is at least about 2-fold, about 4-fold, about 6-fold, about8-fold, about 10-fold, about 20-fold, about 40-fold, about 60-fold,about 80-fold, about 100-fold, about 120-fold, about 140-fold, about160-fold, lower than the K_(D) value for binding of a ROBO1-Fc proteinto SLIT2.

Biological Activity Assays

In certain embodiments, the recombinant ROBO2-Fc protein disclosedherein reduces at least one biological activity of ROBO2-SLIT signaling.Such activity includes, but is not limited to, binding between ROBO2 andSLIT ligand, binding of intracellular signaling molecules (such assrGAP1 or Nck) to the intracellular domain of ROBO2, and/or downstreamactivities of ROBO2-SLIT signaling, such as, actin polymerization,podocyte adhesion, and/or SLIT2-N mediated inhibition of neuronal cellmigration, among other ROBO2-SLIT activities known in the art. Whether arecombinant ROBO2-Fc protein reduces an activity of ROBO2 can beassessed by a number of assays well known in the art. For example,assays known in the art can be used to determine whether the recombinantROBO2-Fc protein: (a) inhibits the binding of SLIT to ROBO2; (b) reducesthe binding of srGAP1 and ROBO2; (c) reduces the binding of Nck andROBO2; (d) inhibits ROBO2-dependent SLIT2-N activity; (e) inhibits actinpolymerization; (f) inhibits podocyte adhesion; and/or (g) inhibitsSLIT2-N mediated inhibition of neuronal cell migration.

In certain embodiments, the recombinant ROBO2-Fc protein inhibits thebinding of SLIT ligand to ROBO2 (e.g., can be assessed by assessingcompetitive binding between the recombinant ROBO2-Fc protein and ROBO2to SLIT2). For example, an assay may compare (i) the binding of ROBO2and SLIT in the presence of the recombinant ROBO2-Fc protein with (ii)the binding of ROBO2 and SLIT in the absence of the recombinant ROBO2-Fcprotein. The reduction in binding of ROBO2 and SLIT can be at leastabout 10%, at least about 20%, at least about 30%, at least about 40%,at least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99%, in thepresence of the recombinant ROBO2-Fc protein compared with binding ofROBO2 and SLIT in the absence of the test recombinant ROBO2-Fc protein.The expected binding of SLIT to ROBO2 in the absence of the recombinantROBO2-Fc protein can be set as 100%.

In certain embodiments, the recombinant ROBO2-Fc protein inhibits thebinding of SLIT to ROBO2, with a half maximal inhibitory concentration(IC₅₀) of not more than about 1×10⁻⁷ M, not more than about 1×10⁻⁸ M,not more than about 1×10⁻⁹ M, not more than about 1×10⁻¹⁰ M, not morethan about 1×10⁻¹¹ M, not more than about 1×10⁻¹² M, not more than about1×10⁻¹³ M, not more than about 1×10⁻¹⁴ M, not more than about 1×10⁻¹⁵ M,from about 1×10⁻⁷ M to about 5×10⁻¹⁴ M, from about 1×10⁻⁷ M to about1×10⁻¹⁴ M, from about 1×10⁻⁷ M to about 5×10⁻¹³ M, from about 1×10⁻⁷ Mto about 1×10⁻¹³ M, from about 1×10⁻⁷ M to about 5×10⁻¹² M, or fromabout 1×10⁻⁷ M to about 1×10⁻¹² M. In some embodiments, the recombinantROBO2-Fc protein has a half maximal inhibitory concentration (IC₅₀) ofnot more than 15 nM, about 13 nM, about 11 nM, about 9 nM, about 7 nM,about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nMas measured by a homogenous time-resolved fluorescence (HTRF) assay forinhibition of binding of ROBO2 to SLIT2. The IC₅₀ may be assessed usinga fragment of SLIT or ROBO2, such as SLIT-N, and Ig domain 1 of ROBO2,or Ig domains 1 & 2 of ROBO2.

The inhibitory activity of a recombinant ROBO2-Fc protein can also beassessed by measuring the level of ROBO2-dependent SLIT-N activity, suchas actin polymerization, podocyte adhesion, and/or SLIT2-N mediatedinhibition of neuronal cell migration. For example, the assay cancompare (i) neuronal cell migration in the presence of ROBO2, SLIT, andthe recombinant ROBO2-Fc protein with (ii) neuronal cell migration inthe presence of ROBO2, SLIT, but in the absence of the recombinantROBO2-Fc protein. The increase in neuronal cell migration can be atleast about 10%, at least about 20%, at least about 30%, at least about40%, at least about 50%, at least about 60%, at least about 70%, atleast about 80%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%, inthe presence of the recombinant ROBO2-Fc protein compared with neuronalcell migration in the absence of the recombinant ROBO2-Fc protein. Thebaseline neuronal cell migration in the absence of the recombinantROBO2-Fc protein can be set as 0%.

In certain embodiments, the recombinant ROBO2-Fc protein inhibitsROBO2-dependent SLIT-N activity, such as actin polymerization, podocyteadhesion, and/or SLIT2-N mediated inhibition of neuronal cell migration,with a half maximal inhibitory concentration (IC₅₀) of not more thanabout 1×10⁻⁷ M, not more than about 1×10⁻⁸ M, not more than about 1×10⁻⁹M, not more than about 1×10⁻¹⁰ not more than about 1×10⁻¹¹ M, not moreM, than about 1×10⁻¹² M, not more than about 1×10⁻¹³ M, not more thanabout 1×10⁻¹⁴ M, not more than about 1×10⁻¹⁵ M, from about 1×10⁻⁷ M toabout 5×10⁻¹⁴ M, from about 1×10⁻⁷ M to about 1×10⁻¹⁴ M, from about1×10⁻⁷ M to about 5×10⁻¹³ M, from about 1×10⁻⁷ M to about 1×10⁻¹³ M,from about 1×10⁻⁷ M to about 5×10⁻¹² M, or from about 1×10⁻⁷ M to about1×10⁻¹² M. In certain embodiments, IC₅₀ of from about 1×10⁻¹⁰ M to about1×10⁻¹³ M is preferred. In certain embodiments, IC₅₀ of from about5×10⁻¹¹ M to about 5×10⁻¹² M is preferred. In some embodiments, therecombinant ROBO-Fc protein has a half maximal inhibitory concentration(IC₅₀) of not more than about 75 nM, about 65 nM, about 55 nM, about 45nM, about 35 nM, about 25 nM, about 15 nM, about 5 nM as assessed bymeasuring SLIT2-N mediated inhibition of neuronal cell migration.

In certain embodiments, the characteristics of the recombinant ROBO-Fcprotein of the invention is further assessed using other biologicalactivity assays, e.g., in order to evaluate its potency, pharmacologicalactivity, and potential efficacy as a therapeutic agent. Such assays areknown in the art and depend on the intended use for the recombinantprotein. Examples include e.g., toxicity assays, immunogenicity assays,stability assays, anti-drug antibody assays, and/or PK/PD profiling.

Nucleic Acids and Methods of Producing Recombinant ROBO2 proteins

The invention also provides polynucleotides encoding the recombinantROBO2 proteins of the invention. The invention also provides a method ofmaking any of the polynucleotides described herein. Polynucleotides canbe made and expressed by procedures known in the art.

In one aspect, the invention provides polynucleotides or compositionscomprising polynucleotides encoding a recombinant ROBO2 proteincomprising a portion of a ROBO2 extracellular domain (ECD) and furthercomprising an immunoglobulin domain, wherein the extracellular domaincomprises: at least two immunoglobulin-like (Ig-like) domains; and aC-terminus sequence consisting of the sequence of SEQ ID NO: 12.

In one aspect, the invention provides polynucleotides or compositions,comprising polynucleotides encoding a recombinant ROBO2 proteincomprising amino acid residues 1 to 203 according the numbering setforth in SEQ ID NO: 1 and further comprising an immunoglobulin domain.

In some embodiments, the invention provides polynucleotides orcompositions, comprising polynucleotides encoding any one of thefollowing recombinant ROBO2 proteins: ROBO2-Fc 2.2 (SEQ ID NO: 1),ROBO2-Fc 2.1 (SEQ ID NO: 2), ROBO2-Fc 2.0 (SEQ ID NO: 3), ROBO2-Fc 1.1(SEQ ID NO: 4), ROBO2-Fc 1.0 (SEQ ID NO: 5), ROBO2-Fc 3.0 (SEQ ID NO:6), ROBO2-Fc 4.0 (SEQ ID NO: 7), and ROBO2-Fc S17T R73Y (SEQ ID NO: 19).In some embodiments, the invention provides polynucleotides orcompositions, comprising polynucleotides encoding ROBO2-Fc 2.2 (SEQ IDNO: 1). In some embodiments, the invention provides polynucleotides orcompositions, comprising polynucleotides encoding ROBO2-Fc 2.1 (SEQ IDNO: 2). In some embodiments, the invention provides polynucleotides orcompositions, comprising polynucleotides encoding ROBO2-Fc 2.0 (SEQ IDNO: 3).

The invention also provides polynucleotides or compositions comprisingthe same, wherein the polynucleotide comprises the sequence of the DNAinsert of the plasmid deposited with the ATCC having ATCC Accession No.PTA-124008.

In another aspect, the invention provides polynucleotides and variantsthereof encoding a recombinant ROBO2-Fc protein, wherein such variantpolynucleotides share at least 70%, at least 75%, at least 80%, at least85%, at least 87%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity to any nucleic aciddisclosed herein such as, but not limited to, a nucleic acid comprisingthe nucleic acid of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and 19 and thenucleic acid sequence of the insert of the plasmid deposited with theATCC having ATCC Accession No. PTA-124008. In some embodiments, suchvariant polynucleotides share at least 95%, sequence identity to anynucleic acid disclosed herein such as, but not limited to, a nucleicacid comprising the nucleic acid of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and19. In some embodiments, such variant polynucleotides share at least96%, sequence identity to any nucleic acid disclosed herein such as, butnot limited to, a nucleic acid comprising the nucleic acid of SEQ IDNOs: 1, 2, 3, 4, 5, 6, 7 and 19. In some embodiments, such variantpolynucleotides share at least 97%, sequence identity to any nucleicacid disclosed herein such as, but not limited to, a nucleic acidcomprising the nucleic acid of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and 19.In some embodiments, such variant polynucleotides share at least 98%,sequence identity to any nucleic acid disclosed herein such as, but notlimited to, a nucleic acid comprising the nucleic acid of SEQ ID NOs: 1,2, 3, 4, 5, 6, 7 and 19. In some embodiments, such variantpolynucleotides share at least 99%, sequence identity to any nucleicacid disclosed herein such as, but not limited to, a nucleic acidcomprising the nucleic acid of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7 and 19.

In another aspect, the invention includes polynucleotides and variantsthereof comprising the nucleic acid sequence set forth in SEQ ID NO: 21.In some embodiments, the invention includes polynucleotides and variantsthereof comprising the nucleic acid sequence set forth in SEQ ID NO: 21,and further comprising a nucleic acid sequence encoding the amino acidsequence set forth in SEQ ID NO:17 or SEQ ID NO: 18. In someembodiments, the nucleic acid sequence encoding the amino acid sequenceset forth in SEQ ID NO: 17 or SEQ ID NO: 18 is N-terminal to the nucleicacid sequence set forth in SEQ ID NO: 21.

In one embodiment, the extracellular domain of ROBO2 and theimmunoglobulin Fc domain are encoded by separate polynucleotides.Alternatively, both the extracellular domain of ROBO2 and theimmunoglobulin Fc domain are encoded by a single polynucleotide.

Polynucleotides complementary to any such sequences are also encompassedby the present disclosure. Polynucleotides may be single-stranded(coding or antisense) or double-stranded, and may be DNA (recombinant,cDNA or synthetic) or RNA molecules. RNA molecules include hnRNAmolecules, which contain introns and correspond to a DNA molecule in aone-to-one manner, and mRNA molecules, which do not contain introns.Additional coding or non-coding sequences may, but need not, be presentwithin a polynucleotide of the present disclosure, and a polynucleotidemay, but need not, be linked to other molecules and/or supportmaterials.

Polynucleotides may comprise a native sequence (i.e., a wild typesequence) or may comprise a non-native (i.e., variant) of such asequence. Polynucleotide variants contain one or more substitutions,additions, deletions and/or insertions such that the SLIT bindingability of the encoded polypeptide is not diminished, relative to anative molecule. The effect on the SLIT binding activity of the encodedpolypeptide may generally be assessed as described herein. In someembodiments, variants exhibit at least about 70% identity, in someembodiments, at least about 80% identity, in some embodiments, at leastabout 90% identity, and in some embodiments, at least about 95% identityto a polynucleotide sequence that encodes a recombinant ROBO2-Fc proteincomprising the native (wild type) sequences of ROBO2 and a Fc domain.

In some embodiments, variants encode a recombinant ROBO2 proteincomprising amino acid residues having at least 20, at least 19, at least18, at least 17, at least 16, at least 15, at least 14, at least 13, atleast 12, at least 11, at least 10, at least 9, at least 8, at least 7,at least 6, at least 5, at least 4, at least 3, at least 2, or at least1 amino acid substitutions of the amino acid residues 1 to 203 accordingthe numbering set forth in SEQ ID NO: 1. In some embodiments, variantsencode a recombinant ROBO2 protein comprising amino acid residues havingat least 5 amino acid substitutions of the amino acid residues 1 to 203according the numbering set forth in SEQ ID NO: 1. In some embodiments,variants encode a recombinant ROBO2 protein comprising amino acidresidues having at least 4 amino acid substitutions of the amino acidresidues 1 to 203 according the numbering set forth in SEQ ID NO: 1. Insome embodiments, variants encode a recombinant ROBO2 protein comprisingamino acid residues having at least 3 amino acid substitutions of theamino acid residues 1 to 203 according the numbering set forth in SEQ IDNO: 1. In some embodiments, variants encode a recombinant ROBO2 proteincomprising amino acid residues having at least 2 amino acidsubstitutions of the amino acid residues 1 to 203 according thenumbering set forth in SEQ ID NO: 1. In some embodiments, variantsencode a recombinant ROBO2 protein comprising amino acid residues havingat least 1 amino acid substitution of the amino acid residues 1 to 203according the numbering set forth in SEQ ID NO: 1. These amounts are notmeant to be limiting, and increments between the recited percentages arespecifically envisioned as part of the disclosure.

Two polynucleotide or polypeptide sequences are said to be “identical”if the sequence of nucleotides or amino acids in the two sequences isthe same when aligned for maximum correspondence as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually50 to about 450, or 100 to about 300, in which a sequence may becompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegAlign® program in the Lasergene® suite of bioinformatics software(DNASTAR®, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O., 1978, A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M.O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M., 1989, CABIOS 5:151-153; Myers, E. W.and Muller W., 1988, CABIOS 4:11-17; Robinson, E. D., 1971, Comb. Theor.11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath,P. H. A. and Sokal, R. R., 1973, Numerical Taxonomy the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA80:726-730.

In some embodiments, the “percentage of sequence identity” is determinedby comparing two optimally aligned sequences over a window of comparisonof at least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Variants may also, or alternatively, be substantially homologous to anative gene, or a portion or complement thereof. Such polynucleotidevariants are capable of hybridizing under moderately stringentconditions to a naturally occurring DNA sequence encoding a recombinantROBO2-Fc protein comprising the native (wild type) sequences of ROBO2and a Fc domain (or a complementary sequence).

Suitable “moderately stringent conditions” include prewashing in asolution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C.-65° C., 5×SSC, overnight; followed by washing twice at 65° C. for 20minutes with each of 2×, 0.5× and 0.2×SSC containing 0.1% SDS.

As used herein, “highly stringent conditions” or “high stringencyconditions” are those that: (1) employ low ionic strength and hightemperature for washing, for example 0.015 M sodium chloride/0.0015 Msodium citrate/0.1% sodium dodecyl sulfate at 50° C.; (2) employ adenaturing agent during hybridization, such as formamide, for example,50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMsodium chloride, 75 mM sodium citrate at 42° C.; or (3) employ 50%formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution,sonicated salmon sperm DNA (50 μg/mL), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2×SSC (sodium chloride/sodiumcitrate) and 50% formamide at 55° C., followed by a high-stringency washconsisting of 0.1×SSC containing EDTA at 55° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a ROBO2-Fc polypeptide comprising an amino acidsequence as described herein. Some of these polynucleotides bear minimalhomology to the nucleotide sequence of any native gene. Nonetheless,polynucleotides that vary due to differences in codon usage arespecifically contemplated by the present disclosure. Further, alleles ofthe genes comprising the polynucleotide sequences provided herein arewithin the scope of the present disclosure. Alleles are endogenous genesthat are altered as a result of one or more mutations, such asdeletions, additions and/or substitutions of nucleotides. The resultingmRNA and protein may, but need not, have an altered structure orfunction. Alleles may be identified using standard techniques (such ashybridization, amplification and/or database sequence comparison).

The present invention also includes codon-optimized polynucleotideswherein the nucleic acid sequence has been optimized to maximizeexpression in a particular cell. In general, codon optimization refersto a process of modifying a nucleic acid sequence for enhancedexpression in the host cells of interest by replacing at least one codon(e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, ormore codons) of the native sequence with codons that are more frequentlyor most frequently used in the genes of that host cell while maintainingthe native amino acid sequence, Various species exhibit particular biasfor certain codons of a particular amino acid. Codon bias (differencesin codon usage between organisms) often correlates with the efficiencyof translation of messenger RNA (mRNA), which is in turn believed to bedependent on, among other things, the properties of the codons beingtranslated and the availability of particular transfer RNA (tRNA)molecules. The predominance of selected tRNAs in a cell is generally areflection of the codons used most frequently in peptide synthesis.Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization. Codon usage tables arereadily available, and these tables can be adapted in a number of ways(e.g., Nakamura, Y., et al. “Codon usage tabulated from theinternational DNA sequence databases: status for the year 2000” Nucl.Acids Res. 28:292 (2000)). Computer algorithms for codon optimizing aparticular sequence for expression in a particular host cell are alsoavailable, such as Gene Forge (Aptagen; Jacobus, Pa.), are alsoavailable. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5,10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding arecombinant ROBO2-Fc protein correspond to the most frequently usedcodon for a particular amino acid.

Thus, in one aspect, the modified nucleic acid sequence provides adetectably greater level of expression of recombinant ROBO2 protein in acell compared with the expression of recombinant ROBO2 protein from thewild type nucleic acid sequence of, e.g., nucleic acid encoding ROBO2-Fc2.2 (SEQ ID NO:21), in an otherwise identical cell. This can be referredto as an “expression optimized” or “enhanced expression” nucleic acid,or simply, as a “modified nucleic acid.”

“Optimized” or “codon-optimized” as referred to interchangeably herein,refers to a coding sequence that has been optimized relative to a wildtype coding sequence (e.g., a coding sequence for human ROBO2 and/orhuman Fc domain) to increase expression of the coding sequence, e.g., byminimizing usage of rare codons, decreasing the number of CpGdinucleotides, removing cryptic splice donor or acceptor sites, removingKozak sequences, removing ribosomal entry sites, and the like.

Examples of modifications include elimination of one or more cis-actingmotifs and introduction of one or more Kozak sequences. In oneembodiment, one or more cis-acting motifs are eliminated and one or moreKozak sequences are introduced.

Examples of cis acting motifs that may be eliminated include internalTATA-boxes; chi-sites; ribosomal entry sites; ARE, INS, and/or CRSsequence elements; repeat sequences and/or RNA secondary structures;(cryptic) splice donor and/or acceptor sites, branch points; and SalI.

In one embodiment, the GC content (e.g., the number of G and Cnucleotides present in a nucleic acid sequence) is enhanced relative towild-type ROBO2 and/or human IgG1 Fc domain gene sequence of the novelROBO2 proteins of the invention. The GC content is preferably at least5%, more preferably, at least 6%, yet more preferably, at least 7%, evenmore preferably, at least 8%, more preferably, at least 9%, even morepreferably, at least 10%, yet more preferably, at least 12%, even morepreferably, at least 14%, yet more preferably, at least 15%, morepreferably, at least 17%, even more preferably, at least 20%, evenfurther preferably, at least 30%, yet more preferably, at least 40%,more preferably, at least 50%, even more preferably, at least 60%, andmost preferably, at least 70% greater than the wild type gene (e.g., SEQID NO:21).

In another embodiment, the GC content is expressed as a percentage of G(guanine) and C (cytosine) nucleotides in the sequence. That is, the GCcontent of the wild type nucleic acid encoding ROBO2-Fc 2.2 (SEQ IDNO:21) is less than the GC content of a codon-optimized nucleic acidsequence encoding ROBO2-Fc 2.2.

In one embodiment, the GC content of a modified nucleic acid of theinvention is greater than the GC content of the wild type nucleic acidencoding ROBO2-Fc 2.2 comprising the nucleic acid sequence of SEQ IDNO:21. One skilled in the art would appreciate, knowing the degeneracyof the nucleic acid code, that irrespective of the sequence of thenucleic acid encoding the protein, the amino acid sequence of ROBO2-Fc2.2 expressed therefrom is, preferably, the amino acid sequence of SEQID NO:1.

It is known that methylation of CpG dinucleotides plays an importantrole in the regulation of gene expression in eukaryotes. Specifically,methylation of CpG dinucleotides in eukaryotes essentially serves tosilence gene expression through interfering with the transcriptionalmachinery. As such, because of the gene silencing evoked by methylationof CpG motifs, the nucleic acids and vectors of the invention having areduced number of CpG dinucleotides will provide for high andlong-lasting transgene expression level.

Potential CpG Islands can be identified using publicly availablesoftware found at, e.g.,http://www.bioinformatics.org/sms2/cpg_islands.html. The CpG Islandssoftware can report potential CpG island regions using the methoddescribed by Gardiner-Garden and Frommer, 1987, J. Mol. Biol.196(2):261-282, among many other methods well-known in the art foridentifying potential CpG islands. The calculation can be performedusing a 200 basepair (bp) window moving across the sequence at 1 bpintervals. CpG islands are defined as sequence ranges where the Obs/Expvalue is greater than 0.6 and the GC content is greater than 50%. Theexpected number of CpG dimers in a window can be calculated as thenumber of ‘C’s in the window multiplied by the number of ‘G’s in thewindow, divided by the window length. Thus, the potential CpG islandspresent in a nucleic acid sequence can be readily determined byinputting the sequence at issue into the window provided by the software(indicated by the instructions to “Paste the raw sequence or one or moreFASTA sequences into the text area below. Input limit is 100000characters.”). CpG islands are often found in the 5′ regions ofvertebrate genes, therefore this program can be used to highlightpotential genes in genomic sequences.

The polynucleotides of this disclosure can be obtained using chemicalsynthesis, recombinant methods, or PCR. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence.

For preparing polynucleotides using recombinant methods, apolynucleotide comprising a desired sequence can be inserted into asuitable vector, and the vector in turn can be introduced into asuitable host cell for replication and amplification, as furtherdiscussed herein. Polynucleotides may be inserted into host cells by anymeans known in the art. Cells are transformed by introducing anexogenous polynucleotide by direct uptake, endocytosis, transfection,F-mating or electroporation. Once introduced, the exogenouspolynucleotide can be maintained within the cell as a non-integratedvector (such as a plasmid) or integrated into the host cell genome. Thepolynucleotide so amplified can be isolated from the host cell bymethods well known within the art. See, e.g., Sambrook et al., 1989.

Alternatively, PCR allows reproduction of DNA sequences. PCR technologyis well known in the art and is described in U.S. Pat. Nos. 4,683,195,4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase ChainReaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vectorand inserting it into a suitable host cell. When the cell replicates andthe DNA is transcribed into RNA, the RNA can then be isolated usingmethods well known to those of skill in the art, as set forth inSambrook et al., 1989, for example.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the host cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing the vector.Suitable examples include plasmids and bacterial viruses, e.g., pUC18,pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19,pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such aspSA3 and pAT28. These and many other cloning vectors are available fromcommercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors are further provided. Expression vectors generallyare replicable polynucleotide constructs that contain a polynucleotideaccording to the disclosure. It is implied that an expression vectormust be replicable in the host cells either as episomes or as anintegral part of the chromosomal DNA. Suitable expression vectorsinclude but are not limited to plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, andexpression vector(s) disclosed in PCT Publication No. WO 87/04462.Vector components may generally include, but are not limited to, one ormore of the following: a signal sequence; an origin of replication; oneor more marker genes; suitable transcriptional controlling elements(such as promoters, enhancers and terminator). For expression (i.e.,translation), one or more translational controlling elements are alsousually required, such as ribosome binding sites, translation initiationsites, and stop codons.

In some embodiments, a vector comprises the nucleic acid molecule setforth in SEQ ID NO: 21. In some embodiments, a vector comprises thenucleic acid molecule set forth in SEQ ID NO: 21 and a nucleic acidsequence encoding the amino acid sequence set forth in SEQ ID NO: 17 orSEQ ID NO: 18. In some embodiments, the nucleic acid sequence encodingthe amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18 isN-terminal to the nucleic acid sequence set forth in SEQ ID NO: 21.

The vectors containing the polynucleotides of interest and/or thepolynucleotides themselves, can be introduced into the host cell by anyof a number of appropriate means, including electroporation,transfection employing calcium chloride, rubidium chloride, calciumphosphate, DEAE-dextran, or other substances; microprojectilebombardment; lipofection; and infection (e.g., where the vector is aninfectious agent such as vaccinia virus). The choice of introducingvectors or polynucleotides will often depend on features of the hostcell.

Exemplary host cells include an E. coli cell, a yeast cell, an insectcell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or amyeloma cell. Preferred host cells include a CHO cell, a Human embryonickidney (HEK) 293 cell, or a Sp2.0 cell, among many cells well-known inthe art.

The host cells may be cultured under conditions which allow expressionof the encoded recombinant ROBO2 protein. In some embodiments, theencoded recombinant ROBO2-Fc protein comprises the ROBO2 leader sequenceset forth in SEQ ID NO: 17 or the Ig leader sequence set forth in SEQ IDNO: 18. In some embodiments, the ROBO2 leader sequence (SEQ ID NO: 17)is cleaved during protein production to produce mature ROBO2-Fc. In someembodiments, the Ig leader sequence (SEQ ID NO: 18) is cleaved duringprotein production to produce mature ROBO2-Fc, e.g., ROBO2-Fc 2.2.

4. Formulations and Uses

The recombinant ROBO2 proteins of the invention can be formulated as apharmaceutical composition. The pharmaceutical composition may furthercomprise a pharmaceutically acceptable carrier, excipient, and/orstabilizer (Remington: The Science and practice of Pharmacy 20th Ed.,2000, Lippincott Williams and Wilkins, Ed. K. E. Hoover), in the form oflyophilized formulation or aqueous solution. As used herein,“pharmaceutically acceptable carrier” or “pharmaceutical acceptableexcipient” includes any material which, when combined with an activeingredient, allows the ingredient to retain biological activity and isnon-reactive with the subject's immune system. Examples include, but arenot limited to, any of the standard pharmaceutical carriers such as aphosphate buffered saline solution, water, emulsions such as oil/wateremulsion, and various types of wetting agents. Preferred diluents foraerosol or parenteral administration are phosphate buffered saline (PBS)or normal (0.9%) saline. Compositions comprising such carriers areformulated by well-known conventional methods (see, for example,Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., MackPublishing Co., Easton, Pa., 1990; and Remington, The Science andPractice of Pharmacy, 20th Ed., Mack Publishing, 2000).

Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations, and may comprise bufferssuch as phosphate, citrate, and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).Pharmaceutically acceptable excipients are further described herein.

Diagnostic Uses

The recombinant ROBO2 proteins of the invention can be used for varioustherapeutic or diagnostic purposes. For example, the recombinant ROBO2proteins of the invention may be used as an affinity purification agent(e.g., for in vitro purification of SLIT ligands, such as SLIT2), as adiagnostic agent (e.g., for detecting expression of a SLIT ligand (e.g.,SLIT2 in specific cells, tissues, or serum). Exemplary diagnostic assaysfor a SLIT ligand, such as SLIT2, may comprise, e.g., contacting asample, obtained from a patient, with a recombinant ROBO2 protein of theinvention, wherein the recombinant ROBO2 protein is labeled with adetectable label or reporter molecule.

The invention encompasses use of the recombinant ROBO2 proteinsdisclosed herein as diagnostic imaging methods for the visualization ofa SLIT ligand, such as SLIT2, in a sample, cell, tissue or patient. Forinstance, the recombinant ROBO2 protein can be conjugated to an imagingagent such that the presence of the recombinant ROBO2 protein can bedetected thereby detecting the presence of a SLIT ligand, such as SLIT2.

Therapeutic Uses

Exemplary therapeutic uses of the recombinant ROBO2 proteins of theinvention include treating a renal disease, such as a glomerulardisease, focal segmental glomerular disease (FSGS). The recombinantROBO2 proteins of the invention may also be used in prophylactictreatment (e.g., administering to a subject who has not exhibited adisease symptom but is susceptible to a renal disease such as aglomerular disease, FSGS).

In another aspect, the invention includes treatment of any disorder,disease or condition mediated by or associated with an increased levelof protein in the urine compared with the level of protein in urine inthe absence of the disease, disorder or condition. Such disease,disorder or condition includes, but is not limited to, lupus nephritis,IgA nephropathy, membranous nephropathy (MN), minimal change disease(MCD), fibrosis (such as liver fibrosis), nonalcoholic steatohepatitis(NASH), proteinuria, albuminuria, glomerulonephritis, diabeticnephropathy, nephrotic syndrome, focal glomerulosclerosis, acute renalfailure, acute tubulointerstitial nephritis, pyelonephritis, renal graftrejection, and reflux nephropathy.

For therapeutic applications, the recombinant ROBO2 proteins of theinvention can be administered to a mammal, especially a human, byconventional techniques, such as intravenously (as a bolus or bycontinuous infusion over a period of time), intramuscularly,intraperitoneally, intra-cerebrospinally, subcutaneously,intra-articularly, intrasynovially, intrathecally, orally, topically, orby inhalation. The recombinant ROBO2 proteins of the invention also canbe suitably administered by intra-tumoral, peri-tumoral, intra-lesional,or peri-lesional routes.

Accordingly, in one aspect, the invention provides a method of reducingthe activity of ROBO2, comprising administering to a subject (e.g., ahuman) in need thereof a therapeutically effective amount of arecombinant ROBO2 protein of the invention.

In another aspect, the invention provides a method of preserving ormodulating podocyte function, comprising administering to a subject(e.g., a human) in need thereof a therapeutically effective amount of arecombinant ROBO2 protein of the invention.

In certain embodiments, the subject suffers from or is susceptible to arenal disease. In certain embodiments, the renal disease is a glomerulardisease. In certain embodiments, the renal disease is FSGS.

In certain embodiments, the subject suffers from or is susceptible tonephropathy.

In another aspect, the invention provides a recombinant ROBO2 protein ofthe invention for use in a method of treatment disclosed herein. Forexample, the invention provides a recombinant ROBO2 protein of theinvention for use in reducing the activity of ROBO2 in a cell, reducingthe activity of ROBO2 in a subject, preserving podocyte function in asubject, modulating podocyte function in a subject, treating aglomerular disease in a subject and treating nephropathy in a subject.

In a further aspect, the invention provides the use of a recombinantROBO2 protein of the invention in the manufacture of a medicament forreducing the activity of ROBO2 in a cell, reducing the activity of ROBO2in a subject, preserving podocyte function in a subject, modulatingpodocyte function in a subject, treating a glomerular disease in asubject and treating nephropathy in a subject.

Dosing and Administration

In certain embodiments, the recombinant ROBO2 protein of the inventionis administered subcutaneously. In certain embodiments, the recombinantROBO2 protein of the invention is administered intravenously.

The pharmaceutical compositions may be administered to a subject in needthereof at a frequency that may vary with the severity of the renaldisease. In the case of prophylactic therapy, the frequency may varydepending on the subject's susceptibility or predisposition to a renaldisease. In some embodiments, the pharmaceutical composition isadministered as a single dose subcutaneously or intravenously. In someembodiments, the pharmaceutical composition is administered as multipledoses subcutaneously or intravenously.

The compositions may be administered to patients in need as a bolus orby continuous infusion. For example, a bolus administration of arecombinant ROBO2 protein may be in an amount of from 0.0025 to 200mg/kg body weight, 0.025 to 0.25 mg/kg, 0.010 to 0.10 mg/kg or 0.10-0.50mg/kg. For continuous infusion, a recombinant ROBO2 protein may beadministered at 0.001 to 200 mg/kg body weight/minute, 0.0125 to 1.25mg/kg/min, 0.010 to 0.75 mg/kg/min, 0.010 to 1.0 mg/kg/min. or 0.10-0.50mg/kg/min for a period of 1-24 hours, 1-12 hours, 2-12 hours, 6-12hours, 2-8 hours, or 1-2 hours.

For administration of recombinant ROBO2 proteins dosage amounts may befrom about 1 mg/kg to about 10 mg/kg, from about 2 mg/kg to about 10mg/kg, from about 3 mg/kg to about 10 mg/kg, from about 4 mg/kg to about10 mg/kg, from about 5 mg/kg to about 10 mg/kg, from about 1 mg/kg toabout 20 mg/kg, from about 2 mg/kg to about 20 mg/kg, from about 3 mg/kgto about 20 mg/kg, from about 4 mg/kg to about 20 mg/kg, from about 5mg/kg to about 20 mg/kg, about 1 mg/kg or more, about 2 mg/kg or more,about 3 mg/kg or more, about 4 mg/kg or more, about 5 mg/kg or more,about 6 mg/kg or more, about 7 mg/kg or more, about 8 mg/kg or more,about 9 mg/kg or more, about 10 mg/kg or more, about 11 mg/kg or more,about 12 mg/kg or more, about 13 mg/kg or more, about 14 mg/kg or more,about 15 mg/kg or more, about 16 mg/kg or more, about 17 mg/kg or more,about 19 mg/kg or more, or about 20 mg/kg or more. The frequency of theadministration would depend upon the severity of the condition.Frequency could range from three times per week to once every two orthree weeks.

Additionally, the compositions may be administered to patients viasubcutaneous injection. For example, a dose of 1 to 200 mg recombinantROBO2 protein can be administered to patients via subcutaneous orintravenous injection administered twice a week, once a week, once everytwo weeks, once every three weeks, once every four weeks, once everyfive weeks, once every six weeks, once every seven weeks, once everyeight weeks, once every nine weeks, once every ten weeks, twice a month,once a month, once every two months, or once every three months.

In certain embodiments, the half-life of the recombinant ROBO2 proteinin human is about 24 hours, about 2 days, about 4 days, about 5 days,about 6 days, about 7 days, about 8 days, about 9 days, about 10 days,about 11 days, about 12 days, about 13 days, about 14 days, about 15days, about 16 days, about 17 days, about 18 days, about 19 days, about20 days, about 21 days, about 22 days, about 23 days, about 24 days,about 25 days, about 26 days, about 27 days, about 28 days, about 29days, about 30 days, from about 5 days to about 40 days, from about 5days to about 35 days, from about 5 days to about 30 days, from about 5days to about 25 days, from about 10 days to about 40 days, from about10 days to about 35 days, from about 10 days to about 30 days, fromabout 10 days to about 25 days, from about 15 days to about 40 days,from about 15 days to about 35 days, from about 15 days to about 30days, or from about 15 days to about 25 days,

In certain embodiments, the pharmaceutical composition is administeredsubcutaneously or intravenously every 2-6 weeks, with a dose from about0.1 mg/kg to about 10 mg/kg, from about 0.5 mg/kg to about 10 mg/kg,from about 1 mg/kg to about 10 mg/kg, from about 1.5 mg/kg to about 10mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 0.1 mg/kg toabout 8 mg/kg, from about 0.5 mg/kg to about 8 mg/kg, from about 1 mg/kgto about 8 mg/kg, from about 1.5 mg/kg to about 8 mg/kg, from about 2mg/kg to about 8 mg/kg, from about 0.1 mg/kg to about 5 mg/kg, fromabout 0.5 mg/kg to about 5 mg/kg, from about 1 mg/kg to about 5 mg/kg,from about 1.5 mg/kg to about 5 mg/kg, from about 2 mg/kg to about 5mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 1.5 mg/kg, about 2.0mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 3.5 mg/kg, about 4.0mg/kg, about 4.5 mg/kg, about 5.0 mg/kg, about 5.5 mg/kg, about 6.0mg/kg, about 6.5 mg/kg, about 7.0 mg/kg, about 7.5 mg/kg, about 8.0mg/kg, about 8.5 mg/kg, about 9.0 mg/kg, about 9.5 mg/kg, or about 10.0mg/kg.

In certain embodiments, the pharmaceutical composition is administeredsubcutaneously or intravenously every 2-6 weeks, with a dose of about3.0 mg/kg. In certain embodiments, the pharmaceutical composition isadministered subcutaneous or intravenously every 2-6 weeks, with a doseof from about 2.0 mg/kg to about 10.0 mg/kg.

In some embodiments, the pharmaceutical composition is administeredsubcutaneously or intravenously weekly or every 2 weeks, with a dose ofabout 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225mg, 250 mg, 275 mg, or 300 mg.

In one exemplary embodiment, the pharmaceutical composition isadministered subcutaneously weekly or every 2 weeks. In certainembodiments, the pharmaceutical composition is administeredsubcutaneously weekly, with a dose of about 2 mg/kg. In certainembodiments, the pharmaceutical composition is administeredsubcutaneously weekly, with a dose of about 150 mg.

In certain embodiments, the pharmaceutical composition is administeredintravenously or subcutaneously every 2-6 weeks, with a dose of about10.0 mg/kg. In certain embodiments, the pharmaceutical composition isadministered subcutaneous or intravenously every 2-6 weeks, with a doseof from about 1.0 mg/kg to about 10.0 mg/kg.

In one exemplary embodiment, the pharmaceutical composition isadministered intravenously every month.

The recombinant ROBO2 protein of the invention can be used asmonotherapy or in combination with other therapies to treat, e.g., arenal disease. Other therapies for treating real disease are well-knownin the art and are not listed herein.

5. Kits

The invention also provides kits or an article of manufacture comprisinga recombinant ROBO2 protein of the invention, and instructions for use.Accordingly, in some embodiments, the disclosure provides a kit or anarticle of manufacture, comprising a container, a composition within thecontainer comprising a recombinant ROBO2 protein, and a package insertcontaining instructions to administer a therapeutically effective amountof the recombinant ROBO2 protein for treatment of a patient in needthereof.

In certain embodiments, the kit can contain both a first containerhaving a dried protein and a second container having an aqueousformulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

The instructions relating to the use of recombinant ROBO2 proteins ofthe invention generally include information as to dosage, dosingschedule, and route of administration for the intended treatment. Thecontainers may be unit doses, bulk packages (e.g., multi-dose packages)or sub-unit doses. Instructions supplied in the kits of the inventionare typically written instructions on a label or package insert (e.g., apaper sheet included in the kit), but machine-readable instructions(e.g., instructions carried on a magnetic or optical storage disk) arealso provided.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). The container may further comprise asecond pharmaceutically active agent.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

6. Biological Deposit

Representative materials of the present invention were deposited in theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209, USA, on Feb. 23, 2017. Vector ROBO2-Fc 2.2 having ATCCAccession No. PTA-124008 comprises a DNA insert encoding SEQ ID NO: 1.The deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit. The deposit will be made available byATCC under the terms of the Budapest Treaty, and subject to an agreementbetween Pfizer Inc. and ATCC, which assures permanent and unrestrictedavailability of the progeny of the culture of the deposit to the publicupon issuance of the pertinent U.S. patent or upon laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and assures availability of the progeny to one determined by the U.S.Commissioner of Patents and Trademarks to be entitled thereto accordingto 35 U.S.C. Section 122 and the Commissioner's rules pursuant thereto(including 37 C.F.R. Section 1.14 with particular reference to 886 OG638).

Pfizer Inc., an Applicant of the present application has agreed that ifa culture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions; the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

EXAMPLES

Exemplary methods and materials are described herein, although methodsand materials similar or equivalent to those described herein can alsobe used in the practice or testing of the present invention. Thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Example 1. Generation of ROBO2-Fc 2.2

Selection of ROBO2-Fc 2.2 Through Design of Multiple ROBO2-Fc FusionProteins

In order to select the optimal ROBO2-Fc ligand trap, multiple proteinswere generated consisting of varying lengths of the ROBO2 extracellulardomain fused to an IgG1 Fc domain through a glycine-serine (GS) linker.Studies have shown that for the ROBO1 and ROBO2 receptors their Ig1domain is sufficient to bind to the D2 leucine rich repeat (LRR) domainof SLIT ligands. ROBO2 contains 5 Ig domains (Ig 1-5). Selectioncriteria first focused on generating a ROBO2-Fc fusion protein with theminimal ROBO2 sequence that could bind SLIT2 and then focused onoptimizing that molecule for recombinant protein expression in HEK 293cells. Initially, 4 DNA constructs were generated for expression ofpolypeptide sequences consisting of the Ig1 domain (ROBO2-Fc 1.0; SEQ IDNO: 5), Ig1 and Ig2 domains (ROBO2-Fc 2.0; SEQ ID NO: 3), Ig1, Ig2, andIg3 domains (ROBO2-Fc 3.0; SEQ ID NO: 6), and Ig1, Ig2, Ig3, and Ig4domains (ROBO2-Fc 4.0; SEQ ID NO: 7) fused to the Fc portion of humanIgG1 through a GS linker (Table 23). Expression of the constructs wasdriven by the Ig leader (SEQ ID NO: 18). The ROBO2-Fc 1.0, 2.0 and 3.0proteins did not bind SLIT2. ROBO2-Fc 4.0 did bind. In order to minimizethe ROBO2 portion of the Fc fusion more proteins were generatedincluding a version with the Ig1 domain comprising at the C-terminal theIg1-Ig2 inter-domain linker (SEQ ID NO: 10) (ROBO2-Fc 1.1; SEQ ID NO:4). Another construct involved including the Ig1 and Ig2 domains and theIg2-Ig3 inter-domain linker (VFER (SEQ ID NO: 12)) (ROBO2-Fc 2.1; SEQ IDNO: 2). Addition of VFER (SEQ ID NO: 12) at the C-terminus of theIg1-Ig2 domains, to create the ROBO2-Fc 2.1 protein, enabled robustbinding to SLIT2 (FIG. 3). ROBO2-Fc 1.1 did not bind SLIT2. Utilizingthe Octet Red, ROBO2-Fc proteins were loaded onto anti human-Fc (AHC)sensors at 10 ug/ml and incubated with 100 nM SLIT2 for 7 minutes andthen the sensors were moved to buffer alone for 640 seconds. ROBO2-Fc4.0 was included as a positive control for binding.

Recombinant protein expression of ROBO2-Fc 2.1 in HEK 293 cells was verypoor (3 μg/ml). In order to increase expression, the ROBO2 pre-Ig1sequence (SEQ ID NO: 8) was added and the ROBO2 leader sequence (SEQ IDNO: 17) was also included to the ROBO2-Fc 2.1 expression construct tocreate the ROBO2-Fc 2.2 expression construct. The ROBO2 leader sequence(SEQ ID NO: 17) is cleaved during protein production to produce matureROBO2-Fc 2.2. Addition of the ROBO2 pre-Ig1 sequence (SEQ ID NO: 8)increased protein expression over 25-fold compared to expression of anotherwise identical expression construct not encoding the pre-Ig1sequence SRLRQEDFP (SEQ ID NO: 8) in an otherwise identical host cell.The increased expression did not affect the biological activity ofROBO2-Fc 2.2 which still bound SLIT2 with high affinity (FIGS. 3-4A-C).

Example 2. Characterization of ROBO2-Fc 2.2 Binding and NeutralizingActivities

ROBO2-Fc 2.2 was screened in numerous assays for SLIT2-N binding,neutralization of SLIT2-N binding, and inhibition of SLITx-N functionalactivity. ROBO2-Fc 2.2 binding to SLIT2 was assessed in two ways; first,by surface plasmon resonance (SPR), and second, using a cell based flowcytometry assay to detect ROBO2-Fc 2.2 binding to cell expressed humanSLIT2-N.

SPR Analysis

Human SLIT2 shares a high level of homology with cynomolgus monkeySLIT2; the overall homology is 99% and homology in the leucine-richrepeat domain 2 (D2) of SLIT2 that contains the ROBO2 binding site is100%. Similar to cynomolgus monkey, human and rat SLIT2 share a highdegree of homology with overall sequence identity and within the D2binding domain being 97%. Binding of ROBO2-FC 2.2 to the N terminalfragment of human and rat SLIT2 which contains the D2 region (SLIT2-N),and the specific SLIT2 D2 domain of human and cynomolgus monkey (100%identical) was assessed by SPR. For each cycle of the kinetic titrationexperiment (FIGS. 4A-4C), the ROBO2-Fc 2.2 molecule was non-covalentlycaptured by Anti-Human Fc (AHFc) attached to a C1 chip, which bound tothe human IgG1 Fc region of ROBO2-Fc 2.2. The captured ROBO2-Fc 2.2 wasthen exposed to varying concentrations of the SLIT2 ligands to monitorassociation and dissociation kinetics. ROBO2-Fc 2.2 was diluted to 2 nM.HBS-EP was used as a diluent. Human/cynomolgus monkey SLIT2-D2, humanSLIT2-N and rat SLIT2-N were diluted to 2 nM in HBS-EPB from their stockconcentrations. An 8 point 2-fold dilution series was performed on theSLIT2 ligands, ranging from 2 nM to 15.6 pM using HBS-EPB as thediluent. ROBO2-Fc 2.2 was captured until a reading of 10-15 RUequivalents and then exposed to titrated amounts of a specific SLIT2analyte, (i.e. human/cynomolgus monkey SLIT2-D2, human SLIT2-N, or ratSLIT2-N). Association of the indicated SLIT2 ligand was followed for120s and dissociation monitored for 180s. The apparent binding affinitywas determined using a simple 1:1 interaction model of the kinetic rateconstants. The K_(D) of ROBO2-Fc 2.2 binding to human/cynomolgus monkeySLIT2-D2 (ROBO2 binding domain, 100% identical) was determined to be0.293 nM (FIG. 4A). The K_(D) of ROBO2-Fc 2.2 binding to human SLIT2-N(Nterminal fragment) was determined to be 0.279 nM (FIG. 4B), and,finally, the K_(D) of ROBO2-Fc 2.2 binding to rat SLIT2-N was determinedto be 0.543 nM (FIG. 4C).

Flow Cytometry Assay

ROBO2-Fc 2.2 was conjugated to Alexa Fluor (AF) 647 according to themanufacturer's instructions. Human embryonic kidney (HEK293)overexpressing human SLIT2-N were re-suspended in buffer containing 5%fetal bovine serum in preparation for staining. To stain cells withROBO2-Fc 2.2-AF647, 2× stocks were prepared and an 11-12 point, 2-folddilution series was made in fluorescence-activated cell sorting (FACS)buffer. Cells and the relevant dilution of ROBO2-Fc 2.2-AF647 werecombined in a 96-well u-bottomed plate and incubated at 4° C. for 45minutes. After incubation, 150 μL of FACS buffer was added per well towash the cells. After washing, cells were resuspended in buffer for dataacquisition on a Fortessa Flow Cytometer. Data was analyzed using FlowJosoftware and are represented as geometric mean fluorescence intensity(Geo MFI) of SLIT2-N expressing HEK293 cells minus the Geo MFI ofcontrol HEK293 cells. ROBO2-Fc 2.2 binds to human SLIT2-N overexpressedon HEK293 cells in a dose-dependent manner with high affinity having anEC₅₀ of 9 nM (FIG. 5).

Homogenous Time Resolved Fluorescence (HTRF) Assay

ROBO2-Fc inhibits binding of a SLIT ligand, such as SLIT2, to a cellularROBO2 receptor likely by acting as a ligand trap. A ROBO2-SLITx-N HTRFdye labeling assay was used to assess the ability of ROBO2-Fc 2.2 toneutralize SLIT ligand binding to human ROBO2. In this assay, terbium(Tb) labeled (donor), SNAP-tagged (SNAP-Tag® New England Biolabs; seealso, Keppler et al., 2003, Nat. Biotechnol. 21:86) human ROBO2expressing HEK293 cells were incubated with 5 nM d2-labeled (acceptor)SLITx-N from various species (prepared using a Cisbio d2 dye labelingkit according to manufacturer's instructions), in the presence oftitrated amounts of ROBO2-Fc 2.2 for 1 hour. After incubation,fluorescence at 665 nm and 620 nm was measured on an Envision multilabelplate reader. The HTRF Ratio was calculated as follows: fluorescence at665 nm/fluorescence at 620 nm×10,000. Maximal signal was defined as theHTRF ratio of Tb-labeled ROBO2 cells with d2-labeled SLIT2-N in theabsence of ROBO2-Fc 2.2, the minimum signal was defined as the HTRFratio of Tb-labeled ROBO2 expressing HEK293 cells only. Neutralizationof 3 different SLIT ligands (SLIT1-N, SLIT2-N and SLIT3-N) from severalspecies, including human, cynomolgus monkey, rabbit, rat and mouse, wereused in the assay. The IC₅₀ for each SLITx-N was determined using an11-point, 4-fold dilution series with a top concentration of 4000 nM.The geometric mean of the IC₅₀ across 3 independent experiments wascalculated and is summarized for all the ligands evaluated (Table 2).

TABLE 2 Summary Table of Results for ROBO-Fc 2.2 Inhibition of theSLITx-N:human ROBO2 HTRF Assay IC₅₀ 95% CI SLIT Ligand Species (GeoMean, nM) (nM) n SLIT1-N Human 5.9 4.4-8.0 3 SLIT1-N Cynomolgus Monkey6.5 4.8-8.9 3 SLIT1-N Rabbit 9.8  6.4-15.0 3 SLIT1-N Rat 8.3  5.5-12.5 3SLIT2-N Human 3.9  1.5-10.6 4 SLIT2-N Rabbit 5.7  2.9-10.9 3 SLIT2-NMouse 5.4 4.6-6.4 3 SLIT3-N Human 5.1 3.5-7.4 3 SLIT3-N CynomolgusMonkey 3.6 2.0-6.4 3 SLIT3-N Rabbit 10.2  7.5-13.9 3 SLIT3-N Rat 3.01.7-5.2 3 SLIT3-N Mouse 4.3 2.2-8.5 3 CI = confidence interval; Geo Mean= geometric mean; HTRF = Homogenous time resolved fluorescence; IC₅₀ =Inhibitory concentration at 50% activity; n = Number of determinations;ROBO2 = Roundabout guidance receptor 2; SLIT = Slit guidance ligand; x =1, 2 or 3.The dose-dependent inhibition of SLIT ligand binding from variousspecies to human ROBO2 by ROBO2-Fc 2.2 as assessed by HTRF. The IC₅₀ wasdetermined using an 11-point, 4-fold dose titration of ROBO-Fc 2.2 inthe HTRF assay against a panel of human, cynomolgus monkey, rabbit, rator mouse SLIT ligands. ROBO2-Fc 2.2 was a potent neutralizer of human,cynomolgus monkey, rabbit and rat SLIT1-N binding to human ROBO2 withIC₅₀s ranging from a low of 5.9 nM (human) to a high of 9.8 nM (rabbit);ROBO2-Fc 2.2 also demonstrated dose dependent inhibition of SLIT2-Nbinding to human ROBO2 with the IC₅₀s ranging from 3.9 nM (human) to 5.7nM (rabbit). ROBO2-Fc 2.2 was a potent neutralizer of SLIT3-N binding tohuman ROBO2 with IC₅₀s ranging from 3.6 nM (cynomolgus monkey) to 10.2nM (rabbit). Finally, ROBO2-Fc 2.2 was a potent neutralizer of bothhuman SLIT2-N:human ROBO2 (FIG. 6A) and rat SLIT2-N:human ROBO2 (FIG.6B) binding with an IC₅₀ of 7 nM or 4 nM, respectively.Neuronal Cell Migration Assay

The final selection screen was functional neutralization ofROBO2-dependent SLIT2-N activity. SLIT2-ROBO2 interactions are keyregulators of axonal migration during development. It is known thatSLIT2 is chemo-repulsive for subventricular zone neurons and that thisactivity is ROBO2 dependent. Neuronal tissue explants from thesubventricular zone (SVZ) of rats were isolated and embedded in acollagen matrix. In the presence of SLIT2-N, neuronal cell migration isinhibited (SVZ assay); tissue explants were incubated in the presence of1 nM SLIT2-N with or without titrated amounts of ROBO2-Fc 2.2. Afterincubation, cells were fixed with 4% paraformaldehyde and stained withHoechst 33342. Wide-field fluorescence images were acquired on theOperetta High Content Imager (Perkin Elmer) with a 10× high NAobjective. Nine fields per well with 5% overlap were taken to capturethe entire center area of the well. A Z-stack for each field wasacquired consisting of 6 planes with 1 μm distance between each plane tocapture the full depth of the tissue explant. Analysis was performed inVolocity software (Perkin Elmer). All fields in each well were stitchedtogether. The area of the tissue explant in the center and each nucleusoutside of the tissue explant were detected by Hoechst 33342 staining.Individual nuclei were counted and the distance of the center of eachnucleus to the closest edge of the tissue explant was measured in μm.The mean migration distance of nuclei in the well was multiplied by thenuclei count to obtain the total migration distance for each well.ROBO2-Fc 2.2 was able to restore neuronal cell migration in adose-dependent manner with an IC₅₀ of 51 nM (FIG. 7). These resultsdemonstrate that ROBO2-Fc 2.2 can not only inhibit the binding of SLIT2to ROBO2, but also has a potent dose-dependent neutralizing effect onthe ROBO2-dependent SLIT2 chemo-repulsive activity for SVZ neurons.

Example 3. In Vivo Effects of Novel ROBO2 Proteins

Passive Heymann's Nephritis is a rat model of nephrotoxic injuryaffecting glomerular podocytes, leading to an increase in proteinuria.This model was used to assess the efficacy of ROBO2-Fc 2.2 to reduceproteinuria. Treatment of rats with ROBO2-Fc 2.2 not only reducedproteinuria but also protected podocyte foot process architecture. Asshown in FIG. 8 and FIG. 9, treatment of rats with either a prophylacticor therapeutic dosing regimen of ROBO2-Fc 2.2 reduced the amount ofproteinuria. Lewis rats were injected with sheep antisera raised againstrat kidney brush border (anti-Fx1a (Probetex Inc, basement membrane andpodocytes). The rats develop an immune response to the sheep sera.Complement activation leads to podocyte effacement and an increase inproteinuria between day 3 and 12 followed by a plateau. This mechanismclosely resembles that found in Membranous Nephropathy whereautoantibodies against the podocyte protein PLA2R (in 70% of cases)cause podocyte effacement and nephrotic range proteinuria followingcomplement engagement. Rats were pretreated 24 hours before theadministration of sheep antisera with a dose range to coverapproximately 50, 90 and 99% (1, 5, and 25 mg/kg) of glomerular ROBO2and every 72 hours thereafter (prophylactic dosing regimen). For thetherapeutic dosing regimen ROBO2-Fc 2.2 was administered on day 6 or day9 (5 or 8 days after administration of sheep antisera), a dose given 24hours prior to the administration of sheep antisera was also included inthe study. The maximal reduction in proteinuria was 45% when treatmentbegan prophylactically (FIG. 8) and a repeated measures ANOVAstatistical analysis confirmed the dose response with a p value lessthan 0.001. Treatment with ROBO2-Fc 2.2 administered on day 0, 6 and 9reduced proteinuria to a similar extent (FIG. 9), 40% maximally and witha p value less than 0.001 by repeated measure ANOVA statistical analysesfor each ROBO2-Fc 2.2 treated group compared to the control antibodytreatment. There was no reduction in immune complex deposition in thekidney as determined by complement IHC scoring, indicating the responsewas due to a podocyte protective effect.

To further provide confidence in the modulation of podocyte function andstructure, quantitative analysis of electron micrographs of podocytesubstructure was performed as described below.

Collection, Sampling, and Sectioning

Full face sample kidneys (one kidney per animal) fixed by immersion (4%formaldehyde/1% glutaraldehyde) were received, trimmed to include justthe cortex, and five samples of each kidney were embedded in epoxyresin. The first embedded sample of each kidney was sectioned. If itcontained three glomeruli the sample was thin sectioned and imaged. Ifthis first sample did not contain glomeruli, the other embedded samplesfrom that kidney were sequentially sectioned and similarly evaluated tofind a sample with three glomeruli.

Viewing and Imaging

Selected kidney samples were digitally imaged using a transmissionelectron microscope (Hitachi H-7100) and a digital CCD camera system(Advanced Microscopy Techniques, Danvers, Mass.). Without repetition,three capillary loops of the first three glomeruli found at 200×magnification, were imaged at 5,000× and 10,000× magnification. Thisresulted in 18 digital images per kidney (i.e., three glomeruli perkidney sample x three areas per glomerulus×2 magnifications). To allowevaluation in a blinded fashion, each image was identified only withstudy number, animal number, sample number, and magnification.

Podocyte Foot Process Width and Slit-Diaphragm Density Measurement

ImageJ software (version 1.47v; National Institutes of Health, Bethesda,Md., USA) was used to manually trace and measure the width of footprocesses adjacent to per unit length of the glomerular basementmembrane (GBM) on high magnification transmission electron microscopyimages. Briefly, measurements were carried out in six rats per group andnine 5000×TEM images from each rat. The podocyte foot process width wasmeasured from one end of the slit diaphragm to the other by drawing aline parallel to the GBM. This was repeated for all the foot processesof the image. These results were then adjusted for scale bar and theharmonic mean for each image was calculated.

Slit diaphragm density: The slit diaphragm density was calculated bycounting the number of slit diaphragms and divided by the total GBMlength spanning the area of these slit diaphragms. Briefly, measurementswere carried out in six rats per group and nine 5000×TEM images fromeach rat. The density was calculated by dividing the number of slitdiaphragms to the GBM length. If more than one GBM area was visible orthe GBM was disconnected due to non-visibility of the foot processes,the above procedure was repeated and average of these measurements wereused to calculate the slit diaphragm density. The distance between slitdiaphragms of interdigitating foot processes was calculated acrossmultiple capillary loops, determining the average foot process width.The foot process width of an effaced podocyte will be larger than thatof a normal uneffaced podocyte.

As shown in FIG. 10A, the foot process width of the ROBO2-Fc 2.2 treatedanimals at the 25 mg/kg dose was significantly shorter that the controlantibody treated animals. These data demonstrate that the reduction ofproteinuria was due to an alteration in podocyte substructure.Additionally, as shown in FIG. 10B, the treatment with ROBO2-Fc 2.2increased the density of slit diaphragms (p value less than 0.01 by twotailed T test) indicating that they were less effaced and protected fromthe glomerular insult.

These results demonstrate that ROBO2-Fc 2.2 is efficacious at inhibitingtwo hallmarks of glomerular disease: proteinuria and diffuse podocyteeffacement. Thus, these results support the use of ROBO2-Fc 2.2 as apotential therapeutic to treat glomerular diseases, such as, but notlimited to, Focal Segmental Glomerulosclerosis (FSGS).

The key pharmacologic properties for ROBO2-Fc 2.2 are summarized inTable 3.

TABLE 3 Summary of Key Pharmacologic Properties of ROBO2-Fc 2.2 AssayPharmacodynamic Activity In Vitro Assays Surface Plasmon Resonance Humanand Cynomolgus KD = 293 ± 70 pM Monkey SLIT2-D2 Human SLIT2-N KD = 279 ±37 pM Rat SLIT2-N KD = 543 ± 72 pM Cell Based Binding Assays (FlowCytometry) Human SLIT2-N EC50 = 8.6 ± 5.0 nM Homogenous Time ResolvedFluorescence Human SLIT2-N IC50 = 7.0 ± 3.9 nM Rat SLIT2-N IC50 = 4.0 ±0.7 nM In Vivo Assay Rat Passive Heymann's Dosing prior to nephrotoxicinjury or Nephritis on Day 6 or 9 (5 or 8 days after injury)significantly reduced proteinuria

In vitro pharmacology studies demonstrated that ROBO2-Fc 2.2 bound withthe same high affinity to human SLIT2-N and the identical human andcynomolgus monkey SLIT2-D2 domain. ROBO2-Fc 2.2 bound to the rat SLIT2-Nwith high affinity as well. ROBO2-Fc 2.2 bound cells expressing humanSLIT2-N in a dose-dependent manner. Additionally, ROBO2-Fc 2.2 potentlyinhibited the binding of rat and human SLIT2-N to human ROBO2 in acell-based binding HTRF assay. In vivo mechanistic, pharmacology, andefficacy studies were also conducted using a rat model of proteinuricglomerular disease. Administration of ROBO2-Fc 2.2, in either aprophylactic or therapeutic dosing regimen, led to a statisticallysignificant reduction of proteinuria in the PHN model. These resultssupport the use of ROBO2-Fc 2.2 to treat glomerular diseases such asFocal Segmental Glomerulosclerosis (FSGS).

Example 4. Characterization of ROBO2-Fc S17T/R73Y Binding andNeutralizing Activities

Flow Cytometry Assay

ROBO2-Fc S17T/R73Y was conjugated to Alexa Fluor (AF) 647 according tothe manufacturer's instructions. Cells overexpressing human SLIT2-N wereresuspended in buffer containing 5% fetal bovine serum in preparationfor staining. To stain cells with ROBO2-Fc S17T/R73Y-AF647, 2× stockswere prepared and an 11-12 point, 2-fold dilution series was made inFACS buffer. Cells and the relevant dilution of ROBO2-Fc S17T/R73Y-AF647were combined in a 96-well u-bottomed plate and placed at 4° C. for 45minutes. After incubation, 150 μL of FACS buffer was added per well towash the cells. After washing, cells were resuspended in buffer for dataacquisition on a Fortessa Flow Cytometer. Data was analyzed using FlowJosoftware and are represented as geometric mean fluorescence intensity(Geo MFI) of SLIT2-N expressing HEK293 cells—the Geo MFI of controlHEK293 cells. ROBO2-Fc S17T/R73Y bound to human SLIT2-N overexpressed onhuman embryonic kidney (HEK293) cells with high affinity having an EC₅₀of 2.5 nM (FIG. 11).

Homogenous Time Resolved Fluorescence (HTRF) Assay

A ROBO2-SLIT2-N homogenous time resolved fluorescence (HTRF) assay wasused to assess the ability of ROBO2-Fc S17T/R73Y to block theinteraction of SLIT2-N and cell expressed ROBO2. In this assay, terbium(Tb) labeled (donor), SNAP-tagged ROBO2 expressing HEK293 cells wereincubated with 5 nM d2-labeled (acceptor) human SLIT2-N in the presenceof titrated amounts of ROBO2-Fc S17T/R73Y for 1 hour per manufacturer'sinstructions (Cisbio HTRF d2 Labeling kit 62D2DPEA and Terbium CryptateLabeling kit 62TBSPEA). After incubation, fluorescence at 665 nm and 620nm was measured on an Envision multilabel plate reader. The HTRF Ratiowas calculated as follows: fluorescence at 665 nm/fluorescence at 620nm×10,000. Maximal signal was defined as the HTRF ratio of Tb-labeledROBO2 cells with d2-labeled SLIT2-N in the absence of ROBO2-Fc S17TR73Y, the minimum signal was defined as the HTRF ratio of Tb-labeledROBO2 expressing HEK293 cells only. ROBO2-Fc S17T R73Y was a potentneutralizer of human SLIT2-N:human ROBO2 binding with an IC₅₀ of 1.4 nM(FIG. 12).

Neuronal Cell Migration Assay

As was done for ROBO2-Fc 2.2, ROBO2-Fc S17T/R73Y was evaluated for theability to reverse SLIT2-N mediated inhibition of neuronal cellmigration in a dose-dependent manner. Neuronal tissue explants from thesubventricular zone (SVZ) of rats were isolated and embedded in acollagen matrix. In the presence of SLIT2-N, neuronal cell migration isinhibited (SVZ assay); tissue explants were incubated in the presence of1 nM SLIT2-N with or without titrated amounts of ROBO2-Fc S17T R73Y.After incubation, cells were fixed with 4% paraformaldehyde and stainedwith Hoechst 33342. Wide-field fluorescence images were acquired on theOperetta High Content Imager (Perkin Elmer) with a 10× high NAobjective. Nine fields per well with 5% overlap were taken to capturethe entire center area of the well. A Z-stack for each field wasacquired consisting of 6 planes with 1 μm distance between each plane tocapture the full depth of the tissue explant. Analysis was performedusing Volocity software (Perkin Elmer). All fields in each well werestitched together. The area of the tissue explant in the center and eachnucleus outside of the tissue explant were detected by Hoechst 33342staining. Individual nuclei were counted and the distance of the centerof each nucleus to the closest edge of the tissue explant was measuredin μm. The mean migration distance of nuclei in the well was multipliedby the nuclei count to obtain the total migration distance for eachwell. ROBO2-Fc S17T/R73Y was able to restore neuronal cell migration ina dose-dependent manner with an IC₅₀ of 11.5 nM (FIG. 13).

These results demonstrate that like ROBO2-Fc 2.2, ROBO2-Fc S17T/R73Y cannot only inhibit the binding of SLIT2 to ROBO2, but also has a potentdose-dependent neutralizing functional effect on the ROBO2-dependentSLIT2 chemo-repulsive activity for SVZ neurons. Thus, these data suggestthat like ROBO2-Fc 2.2, ROBO2-Fc S17T/R73Y can be a potentialtherapeutic that may be useful to treat glomerular diseases involvingROBO2-SLIT interaction, such as, Focal Segmental Glomerulosclerosis(FSGS).

Example 5. Structural Analysis of ROBO2-Fc 2.2

Material Preparation, Crystallization, Data Collection, and StructureDetermination:

Expression and Purification of a ROBO2-His Construct

A ROBO2 construct consisting of the ROBO2 pre-Ig1 sequence (SEQ ID NO:8), Ig1 domain, Ig2 domain and the ROBO2 Ig2-3 inter-domain linker (SEQID NO: 12) with a 6× histidine tag (SEQ ID NO: 25) at the C-terminus(ROBO2-His6 (“His6” disclosed as SEQ ID NO: 25)) was expressed in HEK293cells. The construct was purified through Ni Excel column with imidazolegradient elution. The construct was further purified to homogeneity viasize exclusion chromatography using HiLoad 26/200 Superdex 200 (GEHealthcare).

Crystallization

Crystals of ROBO2-His were obtained under the following condition: 100mM Sodium Citrate pH 4.5, 12% PEG8000, which yielded crystals thatdiffracted to 2.19 Å.

Data Collection

Crystals were transiently cryo-protected and synchrotron data collectionwas performed remotely at Advanced Photon Source (APS). Image frameswere processed using software AutoPROC (Global Phasing Ltd). The databelongs to space group P2₁, with unit cells as follows: a=79.307 Å,b=50.887 Å, c=93.854 Å, α=γ=90°, ρ=114.92, with two copies of the humanROBO2 (Ig1+Ig2) per asymmetric unit.

Structure Determination and Refinement

Molecular Replacement searches using homology model of ROBO1 (Ig1+Ig2,PDB code: 2V9Q) yielded convincing solutions of each component.Refinement was performed using software autoBUSTER (Global Phasing Ltd),and the final R/Rfree factors at 2.19 Å are 0.2119 and 0.2425,respectively, with RMSD of bond 0.010 Å, RMSD of angles 1.19°.

Structural Results and Analysis:

Enhancement of Expression Via the ROBO2 Pre-Ig1 Sequence

In Example 1, it has been demonstrated that inclusion of the wild-typeROBO2 pre-Ig1 sequence SRLRQEDFP (SEQ ID NO: 8) drastically improved thelevel of expression for ROBO2-Fc 2.2. It is evident from the crystalstructure of ROBO2-His6 (“His6” disclosed as SEQ ID NO: 25) that Asp7(D7), Phe8 (F8), and Pro9 (P9) are substantially involved in theinteractions vital for structural integrity of ROBO2's first Ig domain(FIG. 14). Asp7 forms a hydrogen bond with Tyr94, while Phe8 forms adouble bond with Arg36 and Asn93 (Tables 4-5; distance between theneighboring residues is within a distance of 3.8 Å or less). Inaddition, Asp7 (D7), Phe8 (F8), and Pro9 (P9) also involve extensive vander Waals' contacts with neighboring residues, rendering more than 50%of their surface areas buried (Tables 6-9). Without wishing to be boundby any particular theory, these data demonstrate that the ROBO2 pre-Ig1sequence bridges together the two β-sheets of ROBO2's first Ig domainand thereby significantly stabilizes the structural fold of theN-terminal region. Surprisingly, these data suggest that thestabilization of the structural fold mediates the increased expressionof the properly folded ROBO2-Fc 2.2 compared with the expression ofROBO2-Fc constructs lacking the pre-Ig1 sequence.

TABLE 4 Hydrogen bonds involving N-terminal residues of ROBO2 (copy1)Residue_1 Residue_1 # Residue_2 Residue_2 # Distance (Å) ASP 7 TYR 942.73 PHE 8 ARG 36 3.3 PHE 8 ASN 93 2.84 PHE 8 ARG 36 3.01

TABLE 5 Hydrogen bonds involving N-terminal residues of ROBO2 (copy2)Residue_1 Residue_1 # Residue_2 Residue_2 # Distance (Å) ASP 7 TYR 942.58 PHE 8 ARG 36 3.14 PHE 8 ASN 93 2.97 PHE 8 ARG 36 2.79

TABLE 6 Minimum distance interaction table for the N-terminal residuesof ROBO2 (copy1) Residue_1 Residue_1 # Residue_2 Residue_2 # Distance(Å) GLU 6 ARG 36 3.44 ASP 7 ARG 36 3.18 ASP 7 TYR 94 2.73 ASP 7 LEU 953.54 PHE 8 GLY 35 3.55 PHE 8 ARG 36 3.01 PHE 8 GLU 34 3.89 PHE 8 LEU 953.78 PHE 8 ASN 93 2.84 PRO 9 ASN 93 3.71 PRO 9 LEU 95 3.59 PRO 9 ARG 113.82

TABLE 7 Minimum distance interaction table for the N-terminal residuesof ROBO2 (copy2) Residue_1 Residue_1 # Residue_2 Residue_2 # Distance(Å) GLU 6 ARG 36 3.74 ASP 7 ARG 36 3.19 ASP 7 TYR 94 2.58 ASP 7 LEU 953.56 PHE 8 GLY 35 3.4 PHE 8 ARG 36 2.79 PHE 8 GLU 34 3.91 PHE 8 LEU 953.6 PHE 8 ASN 93 2.97 PRO 9 ASN 93 3.85 PRO 9 LEU 95 3.58 PRO 9 ARG 113.75

TABLE 8 N-terminal residue buried surface area (BSA) analysis (copy1)Residue Residue # Complex ASA Free ASA BSA BSA % GLU 6 197.12 218.6921.57 9.86 ASP 7 64.6 148.81 84.21 56.59 PHE 8 82.91 179.66 96.75 53.85PRO 9 80.69 173.68 92.99 53.54

TABLE 9 N-terminal residue buried surface area (BSA) analysis (copy2)Residue Residue # Complex ASA Free ASA BSA BSA % GLU 6 191.1 216.4 25.311.69 ASP 7 59.52 147.69 88.17 59.7 PHE 8 82.87 181.29 98.42 54.29 PRO 978.78 172.08 93.3 54.22Enhancement of Expression Via the ROBO2 Ig2-3 Inter-Domain Linker:

Inclusion of the ROBO2 Ig2-3 inter-domain linker V200-F201-E202-R203(VFER; SEQ ID NO: 12) notably increased the expression level of ROBO2-Fc2.2. Val200 is part of the last β-strand in the Ig2 domain of ROBO2.V200 is deeply encircled by neighboring residues through extensive vander Waals' contacts (Tables 10-11); consequently 85% of its surface areahas been buried (Tables 12-13). In addition to Val200,Phe201-Glu202-Arg203 are also substantially involved in hydrogen bondingand van der Waals' interactions within the region (Tables 10-11, 14-15).Collectively, these data suggest that these four amino acid residueseffectively stabilize the structural fold in the C-terminal region ofROBO2's Ig2 domain (FIG. 15). Without wishing to be bound by anyparticular theory, these data suggest that the stabilization of thestructural fold increases the expression of the properly folded ROBO2-Fc2.2 compared with the decreased expression of ROBO2-Fc constructslacking the Ig2-3 interdomain linker.

TABLE 10 Minimum distance interaction table for the C-terminal residuesof ROBO2 (copy1) Residue_1 Residue_1 # Residue_2 Residue_2 # Distance(Å) VAL 200 ARG 173 3.66 VAL 200 VAL 123 3.17 VAL 200 THR 172 3.33 VAL200 LYS 174 3.28 VAL 200 ALA 125 3.84 PHE 201 LYS 174 3.87 PHE 201 VAL122 3.62 PHE 201 ALA 124 3.54 PHE 201 ALA 125 2.87 PHE 201 VAL 123 2.93GLU 202 LYS 174 3.54 ARG 203 ALA 125 3.62 ARG 203 GLU 127 3.53

TABLE 11 Minimum distance interaction table for the C-terminal residuesof ROBO2 (copy2) Residue_1 Residue_1 # Residue_2 Residue_2 # Distance(Å) VAL 200 ARG 173 3.55 VAL 200 VAL 123 3.27 VAL 200 THR 172 3.27 VAL200 LYS 174 3.88 VAL 200 ALA 125 3.75 PHE 201 VAL 122 3.6 PHE 201 ALA124 3.56 PHE 201 ALA 125 2.84 PHE 201 VAL 123 2.94 GLU 202 LYS 174 3.43ARG 203 ALA 125 3.03 ARG 203 GLU 127 3.12

TABLE 12 C-terminal residue buried surface area (BSA) analysis (copy1)Residue Residue # Complex ASA Free ASA BSA BSA % VAL 200 21.95 207.67185.72 89.43 PHE 201 93.47 167.02 73.55 44.04 GLU 202 138.33 166.7528.42 17.04 ARG 203 96.04 153.35 57.31 37.37

TABLE 13 C-terminal residue buried surface area (BSA) analysis (copy2)Residue Residue # Complex ASA Free ASA BSA BSA % VAL 200 27.85 206.09178.24 86.49 PHE 201 99.37 171.31 71.94 41.99 GLU 202 113.98 153.4539.47 25.72 ARG 203 77.8 159.3 81.5 51.16

TABLE 14 Hydrogen bonds involving C-terminal residues of ROBO2 (copy1)Residue_1 Residue_1 # Residue_2 Residue_2 # Distance (Å) VAL 200 LYS 1743.28 PHE 201 ALA 125 2.87 PHE 201 VAL 123 2.93

TABLE 15 Hydrogen bonds involving C-terminal residues of ROBO2 (copy2)Residue_1 Residue_1 # Residue_2 Residue_2 # Distance (Å) PHE 201 ALA 1252.84 PHE 201 VAL 123 2.94 GLU 202 LYS 174 3.43 ARG 203 ALA 125 3.03 ARG203 GLU 127 3.23 ARG 203 GLU 127 3.12

Example 6. Structural Analysis of ROBO2-Fc S17T/R73Y

Material Preparation, Crystallization, Data Collection, and StructureDetermination:

Expression

The human ROBO2 S17T/R73Y-His6 construct (“His6” disclosed as SEQ ID NO:25) consists of human ROBO2 Ig1 domain with 2 point mutations (S17T andR73Y) followed by a C-terminal His×6 Tag (SEQ ID NO: 25). The humanSLIT2 construct consists of the D2 domain of SLIT2 (271-479) followed bya C-terminal His6 Tag (SEQ ID NO: 25). These constructs were expressedin HEK293 cells separately and the conditioned media was harvested 120hours post transfection.

Purification of Human ROBO2 S17T/R73Y-His6 (“His6” Disclosed as SEQ IDNO: 25) and SLIT2 D2 Domain

Human ROBO2 S17T/R73Y-His6 (“His6” disclosed as SEQ ID NO: 25) waspurified from conditioned media using Nickel Sepharose HP (GEHealthcare) and a gradient of imidazole. The peak fractions werecombined and diluted to 20 mM NaCl, adjusted to pH 6.0 and loaded onto aHiTrap, Q Sepharose Fast Flow (Q FF) column placed in tandem with aHiTrap Strong sulfopropyl (SP). High Performance (HP) column. Afterextensive wash, Q FF column was removed and a gradient of NaCl wasapplied to SP HP column. Fractions were pooled based on extent ofglycosylation and were dialyzed against TBS.

Human SLIT2 D2 domain was captured from conditioned media using NickelSepharose HP (GE Healthcare) and purified with gradient of imidazole.The peak fractions were pooled and further purified through Superdex 200size exclusion chromatography under buffer containing 50 mM Tris HCl pH7.5, 1000 mM NaCl. Fractions containing SLIT2 D2 were pooled andadjusted to 500 mM NaCl.

Complex Formation of Human ROBO2 S17T/R73Y-His and SLIT2 D2 Domain

Due to unexpected interaction between SLIT2 and the resin of the gelfiltration column, the complex of ROBO2 S17T/R73Y-His6 (“His6” disclosedas SEQ ID NO: 25) and SLIT2 could not be obtained through size exclusionchromatography. Instead, purified ROBO2 S17T/R73Y-His6 (“His6” disclosedas SEQ ID NO: 25) was mixed with SLIT2 D2 domain at 1:1 molar ratio andconcentrated for crystallization attempts.

Crystallization

Crystals of ROBO2 S17T/R73Y-His6 (“His6” disclosed as SEQ ID NO: 25) incomplex with SLIT2 D2 domain were obtained in the following condition:100 mM Sodium Citrate pH5.5, 15% PEG6000. It yielded crystals thatdiffracted to 1.78 Å.

Data Collection

Crystals were transiently cryo-protected and synchrotron data collectionwas performed remotely at Advanced Photon Source (APS). Image frameswere processed using software AutoPROC (Global Phasing Ltd). The databelongs to space group P2₁2₁2₁, with unit cells as follows: a=163.251 Å,b=41.896 Å, c=50.938 Å, α=β=γ=90°, with one copy of the complex perasymmetric unit.

Structure Determination and Refinement

Molecular Replacement searches using homology model of ROBO1 Ig1 domainand SLIT2 D2 domain (PDB code: 2V9T) yielded convincing solutions ofeach component. Refinement was performed using software autoBUSTER(Global Phasing Ltd), and the final R/Rfree factors at 1.78 Å are 0.1848and 0.2354, respectively, with RMSD of bond 0.010 Å, RMSD of angles1.10°.

Structural Results and Analysis

The crystal structure of ROBO2 S17T/R73Y-His6-SLIT2 (“His6” disclosed asSEQ ID NO: 25) aligns extensively with that of ROBO1-SLIT2 and theROBO-SLIT interface is highly conserved between ROBO2 and ROBO1.Therefore, the publicly available ROBO1-SLIT2 structure can be used as asubstitute for ROBO2-SLIT2 for structural comparison to probe theimpacts of S17T and R73Y.

Ser17 (S17) of ROBO2 interacts with Arg287 of SLIT2 via a hydrogen bond(FIG. 16). The hydrogen bond with Arginine is conserved regardless ofthe mutation; however, Thr17 provides additional van der Waals' contactwith Arg287, and thus elicits slight energetic advantage over thewild-type Ser17.

R73Y of ROBO2 interacts with Tyr404 of SLIT2 (FIG. 16). The van derWaals' interactions caused by pi-pi stacking of two tyrosine residuesare clearly more superior over those between a tyrosine and a flexiblearginine side-chain. This is evident through the percentage of buriedsurface area (42% for Tyr73 vs. 22% for Arg73; Tables 16-19). The Arg toTyr mutation in ROBO2 S17T/R73Y is likely to be the main contributor forthe increase in affinity compared with wild type ROBO2 comprising S17and R73.

In addition, S17T and R73Y are located at the opposite ends of theROBO2-SLIT2 interface (FIG. 16). Without wishing to be bound by anyparticular theory, stabilizing interactions at these positions likelyhelp ROBO2 to better ‘stick’ with SLIT2, and thus increases globalbinding affinity of the two proteins.

TABLE 16 Interaction table for the key mutations in ROBO2 Residue_1Residue_1 # Chain Residue_2 Residue_2 # Chain Distance (Å) THR 17 ROBO2ARG 287 SLIT2 3.28 TYR 73 ROBO2 TYR 404 SLIT2 3.43

TABLE 17 Interaction table for the corresponding wild-type residues inROBO1 Residue_1 Residue_1 # Chain Residue_2 Residue_2 # Chain Distance(Å) SER 17 ROBO1 ARG 287 SLIT2 3.25 ARG 73 ROBO1 TYR 404 SLIT2 3.01

TABLE 18 Buried surface area analysis for the key mutations in ROBO2Residue Complex Free BSA Residue # Chain ASA ASA BSA % THR 17 ROBO264.76 99.97 35.21 35.22 TYR 73 ROBO2 44.17 75.85 31.68 41.77

TABLE 19 Buried surface area analysis for the corresponding wild-typeresidues in ROBO1 Residue Complex Free BSA Residue # Chain ASA ASA BSA %SER 17 ROBO1 53.39 79.32 25.93 32.69 ARG 73 ROBO1 98.23 125.94 27.71 22

Example 7: Post-Translational Modifications of ROBO2-Fc 2.2

LC/MS—Peptide Mapping

Analysis of post-translational modifications of ROBO2-Fc 2.2 wasaccomplished by peptide mapping. Briefly, ROBO2-Fc 2.2 was reduced,alkylated and digested with the lysine specific protease, LysylEndopeptidase (Lys-C). The Lys-C proteolytic peptides were analyzed byreversed-phase high performance liquid chromatography (RP-HPLC) with UVdetection at 214 nm.

All major and minor peaks in the peptide map for ROBO2-Fc 2.2 wereidentified by on-line electrospray ionization mass spectrometry(RP-HPLC/ESI MS or LC/MS). The observed masses for each peak wereconsistent with the expected Lys-C proteolytic peptides from ROBO2-Fc2.2. These LC/MS-peptide mapping results demonstrate that ROBO2-Fc 2.2contains the correct amino acid sequence, as predicted from the cDNAsequence. LC/MS analysis indicated the presence of N-linkedoligosaccharides in peptide K6 (which was detected as K4K5K6), whichcontains a N¹⁰²AS consensus sequence for N-linked glycosylation. Themajor oligosaccharide structures identified in the K4K5K6 glycopeptideinclude biantennary complex-type structures containing either twogalactose residues with one terminal N-acetylneuraminic acid residue(G2F+1NeuAc) or two terminal N-acetylneuraminic acid residues(G2F+2NeuAc). A minor level K4K5K6 glycopeptide containing two terminalgalactose residues (G2F) was observed. Other minor level speciesrepresenting triantennary and tetraantennary complex-type structureswith core-substituted fucose containing up to four terminalN-acetylneuraminic acid residues were observed.

LC/MS analysis also indicated the presence of N-linked oligosaccharidesin peptide K19 (observed as K18K19), which contains the N²⁹¹ST consensussequence for N-linked glycosylation. The major oligosaccharidestructures identified on K18K19 include asialo-biantennary complex-typestructures containing zero (GOF) or 1 (G1F) terminal galactose residues.Minor level K18K19 with the asialo-biantennary complex-type structurecontaining two galactose residues (G2F) was observed. Minor and tracelevel glycopeptide K18K19 containing truncated N-glycans and complexN-glycans containing up to two terminal N-acetylneuraminic acid residueswere detected.

TABLE 20 LC/MS - N-linked glycosylation Character- istics ResultsN-linked N-linked glycosylation site occupancy confirmed at N²⁹¹STglycosyl- in the K18K19 peptide ation (Major >40%, Minor 5-40%, Trace<5%): N-glycans detected (identities and relative abundances): Major:G0F, G1F Minor: G0, G2F Trace: aglycosylated, G0 minus GlcNAc(truncated), G0F minus GlcNAc (truncated), Man5 (high mannose), G1,G1F + 1NeuAc, G2F + 1NeuAc, and G2F + 2NeuAc N-linked glycosylation siteoccupancy confirmed at N¹⁰²AT in the K4K5K6 and K5K6 peptides in thepeptide map (Major >40%, Minor 5-40%, Trace <5%): N-glycans detected(identities and relative abundances): Major: G2F + 1NeuAc, G2F + 2NeuAcMinor: G2F, G3-TriF + 2NeuAc, G3-TriF + 3NeuAc, G4-TetraF + 3NeuAc, andG2F + 2NeuAc (K5K6 peptide) Trace: G0 minus GlcNAc (truncated), G1F +1NeuAc, G4-TetraF + 2NeuAc, G4 TetraF + 4NeuAc, G2F + 1NeuAc (K5K6peptide)

Example 8: Safety Pharmacology Study

ROBO2-Fc 2.2 was administered once weekly by intravenous (IV) injectionfor 6 weeks (total of 6 doses) at a dose of 50 mg/kg/dose to telemeteredmale monkeys to evaluate cardiovascular (CV) endpoints. Statisticallysignificant differences in heart rate (HR) and activity were observed.However, these differences were small in magnitude, sporadic in nature,and not sustained over time; therefore, they were not consideredROBO2-Fc 2.2 related. IV administration of ROBO2-Fc 2.2 at 50 mg/kg/doseonce weekly for 6 weeks produced no ROBO2-Fc 2.2-related changes inblood pressure (BP), HR, or activity at any time throughout the study.The C_(max) values were 1060, 1030, and 1070 μg/mL on Days 1, 22, and36, respectively.

In a repeat-dose exploratory toxicity study (ETS) in telemetered maleand female cynomolgus monkeys, the effects of once weekly IVadministration of ROBO2-Fc 2.2 at 50 mg/kg/dose for 29 days (total of 5doses) on electrocardiogram (ECG) and blood pressure (BP) parameters andactivity were assessed. There were no ROBO2-Fc 2.2-related effects on CVmeasurements collected through Day 9. On Day 29, combined sex C_(max)was 497 μg/mL.

ECG and HR measurements were incorporated into the Good LaboratoryPractice (GLP)-compliant 3-month repeat-dose toxicity study in male andfemale cynomolgus monkeys at doses of 20, 100, or 300 mg/kg/dose IV, or300 mg/kg/dose subcutaneous (SC). There were no ROBO2-Fc 2.2-relatedeffects on ECG or HR in this study at a mean combined sex C_(max) up to9210 μg/mL. In addition, there were no ROBO2-Fc 2.2-related clinicalobservations that would be suggestive of any respiratory or centralnervous system effects.

In addition, neurofunctional endpoints were assessed in the 3-monthtoxicity study in rats. There were no ROBO2-Fc 2.2-related effects onlocomotor activity or the functional observational battery at doses upto 425 mg/kg/dose IV or at 425 mg/kg/dose SC (mean combined sex C_(max)up to 7610 μg/mL).

Example 9: Pharmacokinetics and Product Metabolism in Animals

Methods of Analysis

I. Quantitation of ROBO2-Fc 2.2 in Rat and Monkey Serum

An electrochemiluminescent (ECL) assay was validated for thequantitation of ROBO2-Fc 2.2 in Wistar Han rat and cynomolgus monkeyserum on the Meso Scale Discovery (MSD®) assay platform (15-1611,15-1609). In these assays, samples containing ROBO2-Fc 2.2 wereincubated onto streptavidin-coated MSD plates coated with biotinylatedanti-ROBO2-Fc antibody capture reagent. The bound ROBO2-Fc 2.2 wasdetected with ruthenylated mouse anti-human IgG Fc antibody. Finaldetection was achieved by adding MSD Read Buffer to produce an ECLsignal that was read using a MSD plate reader. The resulting ECL signalwas directly proportional to the concentration of ROBO2-Fc 2.2. Sampleconcentrations were determined by interpolation from a standard curvethat was fit using a 4PL logistic (autoestimate) model with a weightingfactor of 1/y. The range of quantitation in 100% serum was 50 to 1280ng/mL with a minimum required dilution factor (MRD) of 20×.

II. Detection of Anti ROBO2-Fc 2.2 Antibodies in Rat and Monkey Serum

An ECL assay was validated to detect the presence of anti-drugantibodies (ADA) in Wistar Han rat and cynomolgus monkey serum using theMSD® assay platform. In these assays, biotin and ruthenium-labeledROBO2-Fc 2.2 were co-incubated with study samples, positive controls(anti-ROBO2-Fc 2.2 antibodies in Wistar Han or cynomolgus monkey serum),or negative controls (pooled normal Wistar Han or cynomolgus monkeyserum). Antibodies to ROBO2-Fc 2.2 present in the samples must bind toboth the biotin- and ruthenium-labeled versions of ROBO2-Fc 2.2 to bedetected in these assays. Bound ADA was captured usingstreptavidin-coated MSD plates. Final detection was achieved usingtripropylamine to produce an ECL signal that was measured using a MSDplate reader and reported in relative light units (RLU).

Study samples were tested for ADA using a tiered strategy. Samples wereinitially tested in a screening assay at a minimum required dilution of1:50. Samples that generated an RLU below the assay cutoff point werereported as negative (<1.70, the log 10 of the minimum required dilutionfactor, 50). Samples that generated an RLU at or above the assay cutoffpoint were reanalyzed in a full dilution series to confirm the positiveresult and determine the antibody titer. The antibody titer was definedas the reciprocal dilution of the sample that would have generated anRLU equivalent to the assay cutoff point RLU and the log (to base 10) ofthat titer was reported.

Conclusions regarding the induction of ADA were made based on thecomparison of samples collected prior to dosing on Day 1 and post-dosesample results. If the pre-dose sample tested negative for ADA and thecorresponding post-dose sample tested positive, the animal wasconsidered to be positive for the induction of an ADA response toROBO2-Fc 2.2. If both the pre-dose and post-dose samples tested positivefor ADA, the animal was considered to be positive for the induction ofan ADA response only if the post-dose sample titer was additively atleast 0.48 (log 3, the serial dilution factor) or higher than the titerof the pre-dose sample.

Results

I. Pharmacokinetics

A. Single-Dose Pharmacokinetics

The serum PK of ROBO2-Fc 2.2 were determined in Lewis rats (n=3) andcynomolgus monkeys (n=2) following a single IV dose at 10 mg/kg.ROBO2-Fc 2.2 exhibited a clearance (CL) of 1.5 mL/h/kg in rats, 0.89mL/h/kg in monkey, and a steady state volume of distribution (Vss) of0.19 L/kg in rats, 0.09 L/kg in monkeys, resulting in a terminalhalf-life (t½) of approximately 5 and 8 days in rat and monkey,respectively. Following 10 mg/kg SC administration of ROBO2-Fc 2.2 torats, the estimated bioavailability was 56%.

B. Repeat-Dose Pharmacokinetics (Toxicokinetics; TK)

Rat Toxicokinetics

TK and ADA evaluations were conducted after IV administration of 25,125, or 425 mg/kg/dose, and after SC administration of 425 mg/kg/dose,given once every 3 days to male and female Wistar Han rats (n=6/sex/dosegroup) as part of a 3-month GLP pivotal toxicity study with a 6-weekrecovery phase.

There were no quantifiable concentrations of ROBO2-Fc 2.2 in samplescollected and analyzed prior to dosing on Day 1, or in samples collectedand analyzed from the vehicle control group. Quantifiable concentrationsof ROBO2-Fc 2.2 were observed until Day 88 (last samples collected) anduntil the last day of the recovery phase (Day 135) in the ROBO2-Fc2.2-dosed group.

There were no apparent sex-related differences in systemic exposure (asassessed by maximum concentration [C_(max)] and area under theconcentration curve (AUC) from time 0 to 72 hours [AUC₇₂]) in any dosegroup. Mean systemic exposure increased with increasing dose in anapproximately dose-proportional manner from Day 1 to Day 88. ROBO2-Fc2.2 systemic bioavailability was 24.4% and 25.4% after SC dosing on Days1 and Day 88, respectively. Accumulation ratios (AUC, Day 88/Day 1) were<2.0 for the IV and SC groups (Table 21).

The incidence of ADA induction to ROBO2-Fc 2.2 in the 25, 125 and 425mg/kg IV groups was 0.0% (0/12 animals), 0.0% (0/12 animals), and 0.0%(0/12 animals), respectively, and was 41.7% in the 425 mg/kg SC group(5/12 animals). Serum ROBO2-Fc 2.2 concentrations were similar in theADA-positive animals compared with ADA-negative animals. However, itshould be noted that circulating levels of ROBO2-Fc 2.2 present insamples may have interfered with the detection of ADA.

Cynomolgus Monkey Toxicokinetics

TK and ADA evaluations were conducted after IV administration of 20,100, or 300 mg/kg/dose, and after SC administration of 300 mg/kg/dose,given once every week to male and female cynomolgus monkeys (n=3 or5/sex/dose group) as part of a 3-month GLP pivotal toxicity study with a6-week recovery phase).

There were no quantifiable concentrations of ROBO2-Fc 2.2 in samplescollected and analyzed prior to dosing on Day 1, or in samples collectedand analyzed from the vehicle control group. Quantifiable concentrationsof ROBO2-Fc 2.2 were observed until Day 78 (last samples collected) anduntil the last day of the recovery phase (Day 127 or 128) in theROBO2-Fc 2.2-dosed group.

There were no apparent sex-related differences in systemic exposure (asassessed by C_(max) and AUC₁₆₈) in any dose group. Mean systemicexposure increased with increasing dose in an approximatelydose-proportional manner from Day 1 to Day 78. ROBO2-Fc 2.2 systemicbioavailability (300 mg/kg IV and SC dose) was 53.7% and 77.0% after SCdosing on Day 1 and Day 78, respectively. Accumulation ratios (AUC, Day78/Day 1) were <2.0 for the IV groups, and the ratio was 2.1 for the SCgroup (Table 21).

The incidence of ADA induction to ROBO2-Fc 2.2 in the 20, 100 and 300mg/kg IV groups was 0% (0/6 animals), 0% (0/6 animals), and 20% (2/10animals), respectively, and was 33% in the 300 mg/kg SC group (2/6animals). Serum ROBO2-Fc 2.2 concentrations were similar in theADA-positive animals compared to ADA-negative animals. However, itshould be noted that circulating levels of ROBO2-Fc 2.2 present insamples may have interfered with the detection of ADA.

II. Distribution

The Vss of ROBO2-Fc 2.2 in rats (0.19 L/kg) and monkeys (0.09 L/kg) waslow following a single IV dose, consistent with limited distributioninto extracellular fluids for an Fc containing protein.

III. Pharmacokinetics-Pharmacodynamics and Human PK Predictions

Based on PK/PD modeling (incorporating predicted human PK, measuredROBO2 and SLIT2 serum and kidney concentration data) an estimated weeklyhuman SC dose of 2 mg/kg (150 mg) is predicted to maintain C_(min)target coverage>90% in the kidney. The projected human efficaciousconcentration (C_(eff)) of ˜11 μg/ml serum provides target coverage>90%in the kidney. The projected human steady state C_(max) and AUCtauassociated with a weekly human SC dose of 2 mg/kg (150 mg) are 18.2μg/mL and 2610 μg·h/mL, respectively.

A prediction of human PK for ROBO2-Fc 2.2 has been made by scaling thecynomolgus monkey intravenous PK data based on allometric exponents. Inhuman, ROBO2-Fc 2.2 is predicted to exhibit a clearance of 0.5 mL/h/kgand a steady state volume of distribution of 90 mL/kg providing aterminal half-life of approximately 14 days. The human SCbioavailability is predicted to be 60%.

In initial clinical studies, the exposure limit will be set to a C_(max)of 2930 μg/mL and AUC of 74500 μg·h/mL determined as associated withexposure margins of 161× and 29× the projected human efficaciousexposure, respectively at the projected human efficacious dose (150 mgSC once weekly). This exposure limit is based on the identified noobserved adverse effect level (NOAEL) from the 3-month GLP toxicitystudy in rats.

While no ROBO2-Fc 2.2-related effect on serum cytokine levels was notedin the 3-month GLP toxicity studies in rats or cynomolgus monkeys ETSfollowing administration of ROBO2-Fc 2.2 once weekly at 10 mg/kg/dose SCor 50 or 200 mg/kg/dose IV, respectively, increases of TNF-α and/or IL-6were observed in 1 of 8 human donors and 1 of 12 monkeys in vitro.Although translatability of the in vitro finding is not well understood,measures such as slow administration of ROBO2-Fc 2.2 via IV infusion(with a small fraction of the total dose given in the first hour priorto administration of the remaining dose over the second hour) in singleascending dose phase of the study to allow pause/termination of dosing,close monitoring of vital signs, clinical symptoms and assessment ofcytokine levels will be implemented.

TABLE 21 Mean Overall (Male + Female) Toxicokinetic Parameters ±Standard Deviation^(a) in Rats and Monkeys Species/Study Dose C_(max)T_(max) AUC₇₂ or AUC₁₆₈ AUC₇₂ or AUC₁₆₈/Dose Number (mg/kg/dose)^(b)Route Day (μg/mL) (hours) (μg · h/mL)^(c) ([μg · h/mL]/[mg/kg]) Rat^(d) 25 IV 1 431 0.50 10700 428 16GR156 IV 88 674 0.50 20900 836 (3-month)125 IV 1 2370 0.50 47100 377 IV 88 2930 0.50 74500 596 425 IV 1 92700.50 180000 424 IV 88 7610 0.50 199000 468  425^(e) SC 1 760 48 43900103 SC 88 842 24 50500 119 Cynomolgus Monkey^(f)  20 IV 1  403 ± 48.70.5 ± 0.0 17200 ± 2480  859 ± 124 16GR143 IV 78  470 ± 56.5 0.5 ± 0.024300 ± 3640 1210 ± 182 (3-month) 100 IV 1 2470 ± 246 0.5 ± 0.0  95100 ±15100  951 ± 151 IV 78 2350 ± 503 0.5 ± 0.0 113000 ± 29300 1130 ± 293300 IV 1 7160 ± 565 0.5 ± 0.0 287000 ± 15500  958 ± 51.5 IV 78  9210 ±1080 0.5 ± 0.0 427000 ± 49800 1420 ± 166  300^(g) SC 1 1260 ± 250 32 ±20 155000 ± 29000  515 ± 96.7 SC 78 2340 ± 480 36 ± 29 328000 ± 732001090 ± 244 AUC₁₆₈ = Area under the concentration-time curve from timezero to 168 hours; AUC₇₂ = Area under the concentration-time curve fromtime zero to 72 hours; C_(max) = Maximum observed concentration, IV =Intravenous; SC = Subcutaneous; T_(max) = Time to first occurence ofC_(max). ^(a)Standard deviation not reported for 16GR156 becausenon-serial sampling was used. ^(b)In study 16GR156, animals were dosedonce every 3 days. In study 16GR143, animals were dosed once every week.^(c)AUC₇₂ was used in study 16GR156 and AUC₁₆₈ was used in study16GR143. ^(d)N = 6/sex/dose group (3/sex/time point).^(e)Bioavailability (F %) overall after SC dosing, based on AUC₇₂ was24.4 on Day 1 and 25.4 on Day 88. ^(f)N = 3 or 5/sex/dose group.^(g)Bioavailability (F %) overall after SC dosing, based on AUC₁₆₈ was53.7 on Day 1 and 77.0 on Day 78.

Example 10: Toxicology

ROBO2-Fc 2.2 was administered to rats and cynomolgus monkeys by IV andSC injection, in exploratory (non-Good Laboratory Practice[GLP]-compliant) and pivotal (GLP-compliant) toxicity studies up to 3months (13 weeks) in duration. The no observed adverse effect levels(NOAELs) following 3 months of dosing were:

(i) 125 mg/kg/dose IV (C_(max) of 2930 μg/mL and AUC₇₂ of 74,500μg·h/mL) and 425 mg/kg/dose SC (C_(max) of 842 μg/mL and AUC₇₂ of 50,500μg·h/mL) in rats, and

(ii) 300 mg/kg/dose IV (C_(max) of 9210 μg/mL and AUC₁₆₈ of 427,000μg·h/mL) and SC (C_(max) of 2340 μg/mL and AUC₁₆₈ of 328,000 μg·h/mL) inmonkeys.

I. Repeat-Dose Toxicity

Exploratory and pivotal repeat-dose toxicity studies were conducted withROBO2-Fc 2.2 in rats and cynomolgus monkeys.

A. Rat Study

(i) Exploratory Toxicity Study (ETS)

In an exploratory toxicity study (ETS) in male rats, ROBO2-Fc 2.2 wasadministered once every 3 days for 14 days (5 doses total) at 200mg/kg/dose IV or 10, 50, or 200 mg/kg/dose SC, and was tolerated at alldoses. There were no ROBO2-Fc 2.2-related clinical signs and no changesin body weight or food consumption parameters.

ROBO2-Fc 2.2-related effects included minimal to moderate SCperivascular inflammation in SC injection sites at ≥10 mg/kg/dose SC;higher absolute thymic weights (1.21×-1.44× control) and relative thymicweights (1.17×-1.39× control for organ-to-body weight and 1.22×-1.46×control for organ-to-brain weight) at ≥10 mg/kg/dose SC and IV that werenot associated with a microscopic correlate; higher urine creatinine(1.70× and 1.83× control) and lower urine volume (0.52× and 0.55×control, respectively) at 200 mg/kg/dose SC and IV; higher mean urinespecific gravity (1.011×-1.012× control) at 200 SC and 200 IVmg/kg/dose; and higher mean serum cholesterol (1.38× control) at 200mg/kg/dose IV. The clinical chemistry and organ weight findings were notassociated with any microscopic findings.

(ii) Repeat-Dose Toxicity Study

In a pivotal, repeat-dose rat toxicity study, ROBO2-Fc 2.2 wasadministered once every 3 days for 3 months (31 total doses) at 25, 125,or 425 mg/kg/dose IV or at 425 mg/kg/dose SC to rats, followed by a6-week recovery phase (control and 425 mg/kg/dose IV). Theneurofunctional effects of ROBO2-Fc 2.2 were also evaluated. ROBO2-Fc2.2-related clinical signs included skin lesions (dorsal, thorax,cranial, or injection site) noted in 4/10 male rats at 425 mg/kg/dose SCduring the dosing phase. These changes were not considered adverse asthe incidence was only slightly higher compared with control group (2/15males), and changes were noted only sporadically in the dosing phase andnot present in animals administered ROBO2-Fc 2.2 at doses up to 425mg/kg/dose IV. There were no ROBO2-Fc 2.2-related body weight, foodconsumption, ophthalmology, neurofunctional, or macroscopic findings.

In males, renal findings consisted of minimal glomerulopathy at ≥125mg/kg/dose IV. In females, minimal glomerulopathy was also observed,only at 425 mg/kg/dose IV, and accompanied by minimal to mild tubularbasophilia and hyaline tubular casts, as well as higher urinary protein(100 mg/dL). The morphologic findings of glomerulopathy, tubularbasophilia, and hyaline tubular casts in females were adverse becausethat combination along with non-adverse higher urine protein indicatedimpaired renal function. In males, glomerulopathy was non-adverse,because the finding was much less extensive in distribution, occurred inthe absence of tubular basophilia and hyaline casts, and was notassociated with higher urine protein. Glomerulopathy was characterizedby aggregates of granular eosinophilic material in glomeruli in renalcortices observed by light microscopy and podocyte foot process fusionsurrounding glomerular capillary loops observed by transmission electronmicroscopy. At the end of the recovery phase, glomerulopathy completelyrecovered in males, and partially recovered and was non-adverse infemales. There was also complete recovery of tubular basophilia, hyalinetubular casts, and urinary protein in females. Higher serum totalprotein (1.09×-1.18×) and serum albumin (1.10×-1.20×) were seen in themale group at 425 mg/kg/dose IV, and in the female groups at ≥25mg/kg/dose IV and 425 mg/kg/dose SC. This was observed in the femalegroup at 425 mg/kg/dose IV despite higher urinary protein.

Additional ROBO2-Fc 2.2-related microscopic findings occurred in animalsadministered 425 mg/kg/dose SC, and consisted of an increased incidenceand/or severity (moderate) of hemorrhage and inflammation of theinjection site compared with controls (minimal to mild) and an increasedincidence of minimal lymphoid hyperplasia in the draining lymph node.Increased hemorrhage and inflammation at the SC injection site werenon-adverse because they were only slightly more severe than theconcurrent controls and were not associated with significant tissueinjury beyond what would be expected as a result of the injectionprocedure. The increased incidence of minimal lymphoid hyperplasia inthe draining lymph node was not adverse as it was morphologicallysimilar to controls and likely represented a minor immune response tothe ROBO2-Fc 2.2 and/or the dosing procedure. It is expected that thesefindings would recover once injections were no longer administered.

ROBO2-Fc 2.2-related higher liver weights noted at the conclusion of thedosing phase were non-adverse and consisted of higher mean absolute andrelative to body and brain weights (1.11× to 1.17× compared withcontrol) in female groups at ≥125 mg/kg/dose IV and 425 mg/kg/dose SC.These higher liver weights were non-adverse because of the absence ofcorrelating macroscopic and microscopic findings and absence ofalterations in hepatic enzymes indicative of tissue injury. There wascomplete recovery of higher liver weights.

Additional clinical pathology coagulation and clinical chemistryparameter changes were non-adverse and consisted of higher fibrinogen(1.21×-1.48×) in male and female groups at ≥125 mg/kg/dose IV, highercholesterol (1.37×-2.52×) and triglycerides (1.58×-3.11×) in male groupsat ≥125 mg/kg/dose IV and female groups at ≥25 mg/kg/dose IV and 425mg/kg/dose SC, and higher globulin (1.16×) and higher calcium (1.09×) inthe female group at 425 mg/kg/dose IV. There was complete recovery offibrinogen, cholesterol, triglycerides, total protein, serum albumin,and globulin in male and female groups, but only partial recovery incalcium (1.04× control group at recovery) in the female group at 425mg/kg/dose IV. These clinical pathology changes were non-adverse basedon the small magnitude of the differences between the ROBO2-Fc2.2-administered and control group means and the absence of correlatingmacroscopic and microscopic findings.

A summary of the toxicokinetics in rats following 3 months ofadministration ROBO2-Fc 2.2 can be found in Table 22. Following IVadministration of ROBO2-Fc 2.2 at 25, 125, or 425 mg/kg/dose, or SCadministration at 425 mg/kg/dose once every three days for 3 months (31total doses) to rats, 125 mg/kg/dose IV and 425 mg/kg/dose SC wereidentified as the NOAELs.

B. Monkey Study

(i) Exploratory Toxicity Study (ETS)

In an ETS in male and female cynomolgus monkeys, ROBO2-Fc 2.2 wasadministered once weekly for 29 days (5 total doses) at 50 or 200mg/kg/dose IV or at 10 mg/kg/dose SC. Cardiovascular effects wereevaluated in telemetry-implanted animals in the 50 mg/kg/dose IV group.Selected animals were necropsied on Day 30, the day following the lastdose. Two monkeys from the 50 mg/kg/dose IV group were retained to Day71 for assessment of tolerability and toxicokinetics. Administration ofROBO2-Fc 2.2 was tolerated at all doses. There were no ROBO2-Fc2.2-related effects on survival, clinical signs, body weight, foodconsumption, cardiovascular measurements, in vivo cytokine assessment,hematology and coagulation parameters, and macroscopic and microscopicfindings. ROBO2-Fc 2.2-related effects included minor increases inaspartate aminotransferase in both females at 50 mg/kg/dose IV(1.26×-1.51× baseline) and in the 200 mg/kg/dose IV male and femaleanimals (1.59× and 1.55× baseline, respectively) and minor increases inalanine aminotransferase in the same females at 50 mg/kg/dose IV(1.29×-2.05× baseline) and 200 mg/kg/dose IV (1.29× baseline). Theseenzyme increases were not associated with ROBO2-Fc 2.2-relatedmicroscopic changes in the livers of these animals.

(ii) Repeat-Dose Toxicity Study

In a pivotal repeat-dose cynomolgus monkey toxicity study, ROBO2-Fc 2.2was administered once weekly for 3 months (13 total doses) at 20, 100,or 300 mg/kg/dose IV or at 300 mg/kg/dose SC, followed by a 6-weekrecovery phase (control and 300 mg/kg/dose IV). There were no adverseROBO2-Fc 2.2-related findings in any endpoints evaluated in this study.There were no ROBO2-Fc 2.2-related clinical signs, body weight, foodconsumption, electrocardiogram/heart rate, hematology, coagulation,urinalysis, ophthalmology, organ weight, or macroscopic findings.ROBO2-Fc 2.2-related clinical chemistry alterations included increasedcholesterol (1.25×-1.61× baseline) at 300 mg/kg/dose IV or SC, increasedtriglycerides (1.58×-4.59× baseline) at ≥20 mg/kg/dose IV or 300mg/kg/dose SC, and decreased globulin (0.83×-0.87× baseline) at ≥100mg/kg/dose IV or 300 mg/kg/dose SC. All clinical chemistry alterationswere non-adverse due to their small magnitude of change and absence ofassociated tissue changes. Clinical chemistry alterations fullyrecovered with the exception of increased triglycerides which persistedin the 300 mg/kg/dose IV males following the recovery phase (2.78×-6.79×baseline); quantifiable concentrations of ROBO2-Fc 2.2 were presentuntil Days 128/127 of the recovery phase.

Increased cellularity of lymphoid follicles was observed in the draining(left axillary) and axillary (right axillary) lymph nodes of animalsadministered 300 mg/kg/dose SC. This finding was characterized byminimally to mildly increased size and number of lymphoid follicles withprominent germinal center formation and was consistent with a responseagainst antigenic stimuli. This finding was non-adverse due to itsminimal to mild severity. It is expected that this finding would recoveronce injections were no longer administered. In contrast, no ROBO2-Fc2.2-related microscopic findings were present in animals administeredROBO2-Fc 2.2 at doses up to 300 mg/kg/dose IV. A summary of thetoxicokinetics in cynomolgus monkeys following 3 months ofadministration of ROBO2-Fc 2.2 can be found in Table 22. Followingadministration of ROBO2-Fc 2.2 at 20, 100, or 300 mg/kg/dose IV, or at300 mg/kg/dose SC once weekly for 3 months (13 total doses) to monkeys,300 mg/kg/dose IV and 300 mg/kg/dose SC were identified as the NOAELs.

II. Local Tolerance

IV and SC injection sites were evaluated microscopically in theexploratory and pivotal rat and cynomolgus monkey repeat-dose toxicitystudies. There were no ROBO2-Fc 2.2-related findings in IV injectionsites of rats. ROBO2-Fc 2.2-related findings were only noted in the SCinjections sites of rats. In the rat ETS, minimal to moderatesubcutaneous perivascular inflammation at ≥10 mg/kg/dose SC wasattributed to the physical trauma of injection and considerednon-adverse. In the pivotal rat study, increased severity (moderate) ofhemorrhage and considered non-adverse because they were only slightlymore severe than the concurrent controls and were not associated withsignificant tissue injury beyond what would be expected as a result ofthe injection procedure. Also in the pivotal rat study, ROBO2-Fc2.2-related skin lesions (dorsal, thorax, cranial, or injection site)were noted in males at 425 mg/kg/dose SC during the dosing phase. Thesechanges were not considered adverse as the incidence was only slightlyhigher compared with control group, and were noted sporadically in thedosing phase.

There were no ROBO2-Fc 2.2-related findings in IV injection sites ofcynomolgus monkeys. Hemorrhage and/or neutrophilic inflammation ofvarious seventies present in the IV injection sites in most animals,including controls, in the ETS at doses up to 200 mg/kg/dose IV were notconsidered to be ROBO2-Fc 2.2 related, and there were no ROBO2-Fc2.2-related IV injection site findings in the pivotal cynomolgus monkeystudy at doses up to 300 mg/kg/dose IV. There were no ROBO2-Fc2.2-related findings in SC injection sites of monkeys.

III. Cytokine Release Assays

In an in vitro cytokine release assay (CRA) using human whole bloodsamples, ROBO2-Fc 2.2 elicited tumor necrosis factor-alpha (TNF-α) andinterleukin-6 (IL-6) production in whole blood samples from 1 of 8 humandonors tested. No ROBO2-Fc 2.2-related interferon-gamma (IFN-γ) releasewas observed in human whole blood samples incubated with ROBO2-Fc 2.2.

In an in vitro CRA using cynomolgus monkey whole blood samples collectedprior to the initiation of dosing, ROBO2-Fc 2.2 elicited IL-6 productionin whole blood samples from 1 of 12 cynomolgus monkeys tested, with noROBO2-Fc 2.2-related TNF-α or IFN-γ release observed.

Additionally, blood samples were collected from cynomolgus monkeys inthe ETS following administration of ROBO2-Fc 2.2 once weekly at 10mg/kg/dose SC or 50 or 200 mg/kg/dose IV in order to characterizechanges in cytokine concentrations. There were no ROBO2-Fc 2.2-relatedchanges in serum concentrations of TNF-α, IL-6, or IFN-γ.

IV. C1q and FcγR Binding Assays

ROBO2-Fc 2.2 was evaluated in vitro in a complement protein 1q (C1q)binding ELISA to test its potential to elicit complement-dependentcytotoxicity (CDC), and in a fragment crystallizable gamma receptor(FcγR) binding assay to test its potential for antibody-dependentcell-mediated cytotoxicity (ADCC) activity. ROBO2-Fc 2.2 did not bind toC1q up to the concentrations tested and therefore is considered to havea low potential for inducing CDC. ROBO2-Fc 2.2 binding to all FcγRstested was similar or lower compared with binding seen with the assaycontrol antibody, and was lower compared with data from the positivecontrol antibody. These data suggest that ROBO2-Fc 2.2 has low potentialto elicit ADCC activity.

V. Relationship of Findings to Pharmacokinetics

ROBO2-Fc 2.2 exposure (as assessed by C_(max) and AUC) increased withincreasing dose in an approximately dose-proportional manner afterrepeat IV and SC dosing to rats and cynomolgus monkeys over the doseranges tested. There were no apparent sex-related differences inexposure observed. The threshold concentrations of ROBO2-Fc 2.2associated with key responses and exposure margins calculated againstthese key responses can be found in Table 22.

TABLE 22 Concentrations of ROBO2-Fc 2.2 Associated with Key ResponsesC_(max) AUC_(last) Dose C_(max) ^(a) AUC_(last) ^(a) Exposure ExposureKey Response(s) (mg/kg/day) (μg/mL) (μg · h/mL) Margin^(b) Margin^(b)Repeat-Dose Toxicity Studies 14-Day IV/SC ETS in Male Rats (13MA059;5/group) Target organs: 10 SC 33.7 2040 1.9 <1 SC injection site:perivascular inflammation Lymphoid tissues: ↑ thymic weights Same asabove 50 SC 73.7 3820 4.1 1.5 Same as above, plus 200 SC 245 12500 134.8 Kidney: ↑ urine creatinine, SG, ↓ urine volume Same as above, plus200 IV 6550 68600 360 26 Liver: ↑ CHOL 3-Month IV/SC Toxicity Study inRats With a 6-Week Recovery (16GR156; 10 or 15/sex/group) Target organs:25 IV 674 20900 37 8.0 Liver: ↑: CHOL (F), TRIG (F) Other findings: ↑ALB (F) Kidney: glomerulopathy (M) 125 IV 2930 74500 161 29 Liver: ↑:CHOL (M), TRIG (M), weights (NOAEL) (F) Other findings: ↑ FIB, ↑ TP (F)Same as above, plus 425 IV 7610 199000 418 76 Kidney: glomerulopathy(F), tubular basophilia (F), hyaline casts (F), ↑ urinary protein (F)Liver: ↑: CHOL (M), TRIG (M) Other findings: ↑: GLOB (F), CA (F), FIB,TP (M), ALB (M) Recovery: partial recovery of glomerulopathy and CA (F),complete for all other findings SC injection site: inflammation and 425SC 842 50500 46 19 hemorrhage (NOAEL) Liver: ↑: CHOL (F), TRIG (F),weights (F) Lymphoid tissues: ↑ lymphoid hyperplasia in draining lymphnodes Other findings: ↑ ALB (F), skin lesions (M) 29-Day IV ETS inTelemetered Monkeys With a SC Arm (14MA014; 1 or 2/sex/group) Targetorgans: 50 IV 1050 19100 58 7.3 Liver: ↑: AST (F), ALT (F) Same asabove, plus 200 IV 6420 96200 353 37 Liver: ↑ AST (M) 3-Month IV/SCToxicity Study in Monkeys With a 6-Week Recovery (16GR143; 3 or5/sex/group) Target organs: 20 IV 470 24300 26 9.3 Liver: ↑ TRIG Same asabove, plus 100 IV 2350 113000 129 43 Liver: ↓ GLOB Same as above, plus300 IV 9210 427000 506 164 Liver: ↑ CHOL (NOAEL) Recovery: ↑ TRIG:complete for GLOB and CHOL Lymphoid tissues: ↑ cellularity of lymphoid300 SC 2340 328000 129 126 follicles (NOAEL) Liver: ↑: TRIG, CHOL; ↑GLOB ALB = Albumin; ALT = Alanine aminotransferase; AST = Aspartateaminotransferase; AUC = Area under the concentration-time curve; CHOL =Cholesterol; C_(max) = Maximum (mean) plasma concentration; ETS =Exploratory Toxicity Study; F = Female; FIB = Fibrinogen; GLOB =Globulin; IV = Intravenous; M = Male; NOAEL = No observed adverse effectlevel; SC = Subcutaneous; SG = Specific gravity; TP = Total protein;TRIG = Triglyceride. ^(a)AUC and C_(max) values indicate mean serumconcentrations. Reported values were obtained near termination.^(b)Exposure margins were calculated by dividing C_(max) and AUC_(last)values in animal toxicity studies by the projected human C_(max) of 18.2μg/mL and AUC_(tan) = 2610 μg · h/mL at the projected efficacious humandose of 2 mg/kg [150 mg] SC weekly.

In sum, these data suggest that ROBO2-Fc 2.2 is a potential humantherapeutic for various diseases, disorders and conditions.

The invention thus has been disclosed broadly and illustrated inreference to representative embodiments described above. Those skilledin the art will recognize that various modifications can be made to thepresent invention without departing from the spirit and scope thereof.All publications, patent applications, and issued patents, are hereinincorporated by reference to the same extent as if each individualpublication, patent application or issued patent were specifically andindividually indicated to be incorporated by reference in its entirety.Definitions that are contained in text incorporated by reference areexcluded to the extent that they contradict definitions in thisdisclosure.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

It is specifically contemplated that any limitation discussed withrespect to one embodiment of the invention may apply to any otherembodiment of the invention. Furthermore, any composition of theinvention may be used in any method of the invention, and any method ofthe invention may be used to produce or to utilize any composition ofthe invention. In particular, any aspect of the invention described inthe claims, alone or in combination with one or more additional claimsand/or aspects of the description, is to be understood as beingcombinable with other aspects of the invention set out elsewhere in theclaims and/or description and/or sequence listings and/or drawings.

In so far as specific examples found herein do not fall within the scopeof an invention, said specific example may be explicitly disclaimed.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein thespecification, “a” or “an” may mean one or more, unless clearlyindicated otherwise. As used herein in the claim(s), when used inconjunction with the word “comprising”, the words “a” or “an” may meanone or more than one. As used herein “another” may mean at least asecond or more. Unless otherwise defined herein, scientific andtechnical terms used in connection with the present invention shall havethe meanings that are commonly understood by those of ordinary skill inthe art. Further, unless otherwise required by context, singular termsshall include pluralities and plural terms shall include the singular.The words “comprises/comprising” and the words “having/including” whenused herein with reference to the present invention are used to specifythe presence of stated features, integers, steps or components but doesnot preclude the presence or addition of one or more other features,integers, steps, components or groups thereof.

Although the disclosed teachings have been described with reference tovarious applications, methods, and compositions, it will be appreciatedthat various changes and modifications can be made without departingfrom the teachings herein and the claimed invention below. The examplesare provided to better illustrate the disclosed teachings and are notintended to limit the scope of the teachings presented herein. While thepresent teachings have been described in terms of these exemplaryembodiments, numerous variations and modifications of these exemplaryembodiments are possible without undue experimentation. All suchvariations and modifications are within the scope of the currentteachings.

Where aspects or embodiments of the invention are described in terms ofa Markush group or other grouping of alternatives, the present inventionencompasses not only the entire group listed as a whole, but each memberof the group individually and all possible subgroups of the main group,but also the main group absent one or more of the group members. Thepresent invention also envisages the explicit exclusion of one or moreof any of the group members in the claimed invention.

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated byreference in their entirety. In the event that one or more of theincorporated literature and similar materials differs from orcontradicts this application, including but not limited to definedterms, term usage, described techniques, or the like, this applicationcontrols.

The description and examples detail certain specific embodiments of theinvention and describes the best mode contemplated by the inventors. Itwill be appreciated, however, that no matter how detailed the foregoingmay appear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

TABLE 23 SEQUENCES SEQ DESCRIPTION SEQUENCE  1 ROBO2-Fc 2.2SRLRQEDFPPRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG  2 ROBO2-Fc 2.1PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  3 ROBO2-Fc 2.0PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  4 ROBO2-Fc 1.1PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  5 ROBO2-Fc 1.0PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK 6 ROBO2-Fc 3.0 PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERPTFLRRPINQVVLEEEAVEFRCQVQGDPQPTVRWKKDDADLPRGRYDIKDDYTLRIKKTMSTDEGTYMCIAENRVGKMEASATLTGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  7 ROBO2-Fc 4.0PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERPTFLRRPINQVVLEEEAVEFRCQVQGDPQPTVRWKKDDADLPRGRYDIKDDYTLRIKKTMSTDEGTYMCIAENRVGKMEASATLTVRAPPQFVVRPRDQIVAQGRTVTFPCETKGNPQPAVFWQKEGSQNLLFPNQPQQPNSRCSVSPTGDLTITNIQRSDAGYYICQALTVAGSILAKAQLEVTGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK  8 ROBO2 pre-Ig1 SRLRQEDFPsequence  9 ROBO2 Ig1 PRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVCVARNYLGEA VSRNASLE 10 ROBO2 Ig1-2VALLR linker 11 ROBO2 Ig2 DDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELT 12 ROBO2 Ig2-3 VFER linker13 ROBO2 Ig3 PTFLRRPINQVVLEEEAVEFRCQVQGDPQPTVRWKKDDADLPRGRYDIKDDYTLRIKKTMSTDEGTYMCIAENRVGKMEASATLT 14 ROBO2 Ig4VRAPPQFVVRPRDQIVAQGRTVTFPCETKGNPQPAVFWQKEGSQNLLFPNQPQQPNSRCSVSPTGDLTITNIQRSDAGYYICQALTVA GSILAKAQLEVT 15 GS LinkerGGSGGS 16 IgG1 Fc 3mut EPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK 17ROBO2 Leader MSLLMFTQLLLCGFLYVRVDG 18 Ig Leader MGWSCIIFLVATAGAHS 19ROBO2-Fc SRLRQEDFPPRIVEHPTDVIVSKGEPTTLNCKAEGRPTPTIEWYK S17T/R73YDGERVETDKDDPRSHRMLLPSGSLFFLYIVHGRRSKPDEGSYVCVARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERGGSGGSEPKSSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKVNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 20 ROBO2 Ig1 S17TPRIVEHPTDVIVSKGEPTTLNCKAEGRPTPTIEWYKDGERVETDK R73YDDPRSHRMLLPSGSLFFLYIVHGRRSKPDEGSYVCVARNYLGEA VSRNASLE 21 ROBO2-Fc 2.2TCGCGTCTTCGCCAGGAGGACTTTCCCCCGCGGATTGTGGAGCATCCTTCCGATGTCATCGTCTCTAAGGGCGAGCCCACGACTCTGAACTGCAAGGCGGAGGGCCGGCCAACGCCCACCATTGAGTGGTACAAAGATGGGGAGCGAGTGGAGACTGACAAGGACGATCCCCGGTCCCACAGGATGCTTCTGCCCAGCGGATCCTTATTCTTCTTGCGCATCGTGCACGGGCGCAGGAGTAAACCTGATGAAGGAAGCTACGTTTGTGTTGCGAGGAACTATCTTGGTGAAGCAGTGAGTCGAAATGCGTCTCTGGAAGTGGCATTGTTACGAGATGACTTCCGACAAAACCCCACAGATGTTGTAGTGGCAGCTGGAGAGCCTGCAATCCTGGAGTGCCAGCCTCCCCGGGGACACCCAGAACCCACCATCTACTGGAAAAAAGACAAAGTTCGAATTGATGACAAGGAAGAAAGAATAAGTATCCGTGGTGGAAAACTGATGATCTCCAATACCAGGAAAAGTGATGCAGGGATGTATACTTGTGTTGGTACCAATATGGTGGGAGAAAGGGACAGTGACCCAGCAGAGCTGACTGTCTTTGAACGAGGCGGCAGCGGCGGCAGCGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA GAAGAGCCTCTCCCTGTCCCCCGGA 22Peptidyl linker [Gly-Gly-Ser]_(n) n = 1, 2, 3, 4, 5, or 6 23Peptidyl Linker [Gly-Gly-Gly-Gly-Ser]_(n), n = 1, 2, 3, 4, 5, or 6 24Human ROBO2 MSLLMFTQLLLCGFLYVRVDG (ROBO2 LeaderSRLRQEDFPPRIVEHPSDVIVSKGEPTTLNCKAEGRPTPTIEWYK sequenceDGERVETDKDDPRSHRMLLPSGSLFFLRIVHGRRSKPDEGSYVC underlined)VARNYLGEAVSRNASLEVALLRDDFRQNPTDVVVAAGEPAILECQPPRGHPEPTIYWKKDKVRIDDKEERISIRGGKLMISNTRKSDAGMYTCVGTNMVGERDSDPAELTVFERPTFLRRPINQVVLEEEAVEFRCQVQGDPQPTVRWKKDDADLPRGRYDIKDDYTLRIKKTMSTDEGTYMCIAENRVGKMEASATLTVRAPPQFVVRPRDQIVAQGRTVTFPCETKGNPQPAVFWQKEGSQNLLFPNQPQQPNSRCSVSPTGDLTITNIQRSDAGYYICQALTVAGSILAKAQLEVTDVLTDRPPPIILQGPANQTLAVDGTALLKCKATGDPLPVISWLKEGFTFPGRDPRATIQEQGTLQIKNLRISDTGTYTCVATSSSGETSWSAVLDVTESGATISKNYDLSDLPGPPSKPQVTDVTKNSVTLSWQPGTPGTLPASAYIIEAFSQSVSNSWQTVANHVKTTLYTVRGLRPNTIYLFMVRAINPQGLSDPSPMSDPVRTQDISPPAQGVDHRQVQKELGDVLVRLHNPVVLTPTTVQVTWTVDRQPQFIQGYRVMYRQTSGLQATSSWQNLDAKVPTERSAVLVNLKKGVTYEIKVRPYFNEFQGMDSESKTVRTTEEAPSAPPQSVTVLTVGSYNSTSISVSWDPPPPDHQNGIIQEYKIWCLGNETRFHINKTVDAAIRSVIIGGLFPGIQYRVEVAASTSAGVGVKSEPQPIIIGRRNEVVITENNNSITEQITDVVKQPAFIAGIGGACWVILMGFSIWLYWRRKKRKGLSNYAVTFQRGDGGLMSNGSRPGLLNAGDPSYPWLADSWPATSLPVNNSNSGPNEIGNFGRGDVLPPVPGQGDKTATMLSDGAIYSSIDFTTKTSYNSSSQITQATPYATTQILHSNSIHELAVDLPDPQWKSSIQQKTDLMGFGYSLPDQNKGNNGGKGGKKKKNKNSSKPQKNNGSTWANVPLPPPPVQPLPGTELEHYAVEQQENGYDSDSWCPPLPVQTYLHQGLEDELEEDDDRVPTPPVRGVASSPAISFGQQSTATLTPSPREEMQPMLQAHLDELTRAYQFDIAKQTWHIQSNNQPPQPPVPPLGYVSGALISDLETDVADDDADDEEEALEIPRPLRALDQTPGSSMDNLDSSVTGKAFTSSQRPRPTSPFSTDSNTSAALSQSQRPRPTKKHKGGRMDQQPALPHRREGMTDEEALVPYSKPSFPSPGGHSSSGTASSKGSTGPRKTEVLRAGHQRNASDLLDIGYMGSNSQGQFTGEL

TABLE 24 SEQUENCE ID ASSIGNMENTS ROBO2-Fc Component ROBO2-Fc FullSRLRQEDFP Ig1-2 Ig2-3 GS IgG1 Construct Construct Leader (SEQ ID NO: 8)Ig1 Linker Ig2 Linker Ig3 Ig4 Linker Fc 3mut ROBO2-Fc 5 18 X 9 X X X X X15 16 1.0 ROBO2-FC 4 18 X 9 10 X X X X 15 16 1.1 ROBO2-FC 3 18 X 9 10 11X X X 15 16 2.0 ROBO2-FC 2 18 X 9 10 11 12 X X 15 16 2.1 ROBO2-FC 1 17 89 10 11 12 X X 15 16 2.2 ROBO2-FC 6 18 X 9 10 11 12 13 X 15 16 3.0ROBO2-FC 7 18 X 9 10 11 12 13 14 15 16 4.0 ROBO2-FC 19 17 8 20 10 11 12X X 15 16 S17T R73Y X = COMPONENT NOT PART OF CONSTRUCT

The invention claimed is:
 1. A recombinant Roundabout Receptor 2 (ROBO2)protein comprising (i) a portion of ROBO2 extracellular domain, and (ii)an immunoglobulin domain, wherein said portion of ROBO2 extracellulardomain consists of amino acid residues 1 to 203 according to thenumbering of SEQ ID NO:
 1. 2. The recombinant ROBO2 protein of claim 1,wherein said immunoglobulin domain is a Fc domain of an IgA₁ IgA₂, IgD,IgE, IgM, IgG₁, IgG₂, IgG₃, or IgG₄.
 3. The recombinant ROBO2 protein ofclaim 2, wherein said Fc domain is the Fc domain of human IgG₁.
 4. Therecombinant ROBO2 protein of claim 3, wherein said human IgG₁ Fc domaincomprises L234A, L235A, and G237A substitutions (Eu numbering), and doesnot comprise K447 (Eu numbering).
 5. The recombinant ROBO2 protein ofclaim 4, wherein said Fc domain comprises amino acid residues 210 to 440according to the numbering of SEQ ID NO:
 1. 6. The recombinant ROBO2protein of claim 1, wherein said amino acid residues 1 to 203 accordingto the numbering of SEQ ID NO: 1 are contiguous with said immunoglobulindomain.
 7. The recombinant ROBO2 protein of claim 1, wherein said aminoacid residues 1 to 203 according to the numbering of SEQ ID NO: 1 areconnected via a linker to said immunoglobulin domain.
 8. The recombinantROBO2 protein of claim 7, wherein said linker is a peptidyl linkercomprising about 1 to 30 amino acid residues.
 9. The recombinant ROBO2protein of claim 8, wherein said peptidyl linker is selected from thegroup consisting of: a) a glycine rich peptide; b) a peptide comprisingglycine and serine; c) a peptide having a sequence [Gly-Gly-Ser]_(n),wherein n is 1, 2, 3, 4, 5, or 6 (SEQ ID NO: 22); and d) a peptidehaving a sequence [Gly-Gly-Gly-Gly-Ser]_(n), wherein n is 1, 2, 3, 4, 5,or 6 (SEQ ID NO: 23).
 10. The recombinant ROBO2 protein of claim 9,wherein said peptidyl linker is [Gly-Gly-Ser]₂ (SEQ ID NO: 15).
 11. Therecombinant ROBO2 protein of claim 1, wherein said protein bindsfull-length SLIT2 with a dissociation constant (K_(D)) of about 300 pM,or about 250 pM.
 12. The recombinant ROBO2 protein of claim 11, whereinsaid K_(D) is measured by surface plasmon resonance (SPR).
 13. Therecombinant ROBO2 protein of claim 11, wherein said K_(D) is measured bybio-layer interferometry (BLI), optionally using an eight-channel BLIdetection instrument.
 14. The recombinant ROBO2 protein of claim 1,wherein said protein inhibits binding of SLIT and ROBO2 and/or inhibitsROBO2-dependent SLIT-N activity.
 15. The recombinant ROBO2 protein ofclaim 1, wherein said protein has a half maximal inhibitoryconcentration (IC₅₀) of about 11 nM, about 9 nM, about 7 nM, about 6 nM,about 5 nM, about 4 nM, about 3 nM, about 2 nM, or about 1 nM, asmeasured by a homogenous time-resolved fluorescence (HTRF) assay forinhibition of binding of ROBO2 to SLIT2.
 16. The recombinant ROBO2protein of claim 1, wherein said protein has a half maximal inhibitoryconcentration (IC₅₀) of about 55 nM, as assessed by measuring SLIT2-Nmediated inhibition of neuronal cell migration.
 17. The recombinantROBO2 protein of claim 1, wherein said protein binds a N-terminalcleavage product of human SLIT2 (SLIT2-N) with a dissociation constant(K_(D)) of about 300 pM, or about 250 pM.
 18. The recombinant ROBO2protein of claim 17, wherein said K_(D) is measured by surface plasmonresonance (SPR).
 19. The recombinant ROBO2 protein of claim 17, whereinsaid K_(D) is measured by bio-layer interferometry (BLI), optionallyusing an eight-channel BLI detection instrument.
 20. The recombinantROBO2 protein of claim 1, wherein said protein binds a N-terminalcleavage product of rat SLIT2 (SLIT2-N) with a dissociation constant(K_(D)) of about 600 pM or about 500 pM.
 21. The recombinant ROBO2protein of claim 1, wherein said protein binds a cleavage product ofSLIT2 that comprises the D2 leucine rich repeat domain (SLIT2-D2) with adissociation constant (K_(D)) of about 300 pM, or about 250 pM.
 22. Therecombinant ROBO2 protein of claim 21, wherein said K_(D) is measured bysurface plasmon resonance (SPR).
 23. The recombinant ROBO2 protein ofclaim 21, wherein said K_(D) is measured by bio-layer interferometry(BLI), optionally using an eight-channel BLI detection instrument. 24.An isolated nucleic acid molecule, comprising a nucleotide sequenceencoding the recombinant ROBO2 protein of claim
 1. 25. The isolatednucleic acid molecule of claim 24, wherein said molecule comprises thenucleic acid sequence of SEQ ID NO:
 21. 26. A vector comprising thenucleic acid molecule of claim
 24. 27. A host cell comprising thenucleic acid molecule of claim
 24. 28. A method of making a recombinantROBO2 protein comprising culturing the host cell of claim 27 underconditions wherein the recombinant ROBO2 protein is expressed.
 29. Themethod of claim 28, further comprising isolating the recombinant ROBO2protein.
 30. A pharmaceutical composition comprising the recombinantROBO2 protein of claim 1, and a pharmaceutically acceptable carrier orexcipient.
 31. A method of treating renal disease, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the recombinant ROBO2 protein of claim
 1. 32. The method ofclaim 31, wherein said renal disease is a glomerular disease, FocalSegmental Glomerular Sclerosis (FSGS), or nephropathy.
 33. A recombinantRoundabout Receptor 2 (ROBO2)-Fc protein comprising the amino acidsequence of SEQ ID NO:
 1. 34. The recombinant ROBO2 protein of claim 33,comprising the amino acid sequence encoded by the insert of the plasmiddeposited at the ATCC and having ATCC Accession No. PTA-124008.
 35. Amethod of treating renal disease, comprising administering to a subjectin need thereof a therapeutically effective amount of the recombinantROBO2 protein of claim
 33. 36. The method of claim 35, wherein saidrenal disease is a glomerular disease, Focal Segmental GlomerularSclerosis (FSGS), or nephropathy.
 37. A method of treating FocalSegmental Glomerular Sclerosis (FSGS), comprising administering to asubject in need thereof a therapeutically effective amount of therecombinant ROBO2 protein of claim
 33. 38. A recombinant RoundaboutReceptor 2 (ROBO2)-Fc protein comprising an amino acid sequence at least95% identical to the amino acid sequence of SEQ ID NO:
 1. 39. Arecombinant Roundabout Receptor 2 (ROBO2) protein consisting of theamino acid sequence of SEQ ID NO:
 1. 40. A method of treating renaldisease, comprising administering to a subject in need thereof atherapeutically effective amount of the recombinant ROBO2 protein ofclaim
 39. 41. The method of claim 40, wherein said renal disease is aglomerular disease, Focal Segmental Glomerular Sclerosis (FSGS), ornephropathy.
 42. A method of treating Focal Segmental GlomerularSclerosis (FSGS), comprising administering to a subject in need thereofa therapeutically effective amount of the recombinant ROBO2 protein ofclaim
 39. 43. A pharmaceutical composition comprising the recombinantROBO2 protein of claim 39, and a pharmaceutically acceptable carrier orexcipient.
 44. An isolated nucleic acid molecule, comprising anucleotide sequence encoding a recombinant ROBO2 protein, wherein saidnucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:21.
 45. A vector comprising the nucleic acid molecule of claim
 44. 46. Ahost cell comprising the nucleic acid molecule of claim
 44. 47. A methodof making a recombinant ROBO2 protein comprising culturing the host cellof claim 46 under conditions wherein the recombinant ROBO2 protein isexpressed.
 48. The method of claim 47, further comprising isolating therecombinant ROBO2 protein.